U.S. patent application number 14/124884 was filed with the patent office on 2014-08-28 for culture media for stem cells.
This patent application is currently assigned to Koninklijke Nederlandse Akademie van Wetenschappen. The applicant listed for this patent is Johannes C. Clevers, Meritxell Huch Ortega, Wouter Richard, Toshiro Sato. Invention is credited to Johannes C. Clevers, Meritxell Huch Ortega, Wouter Richard, Toshiro Sato.
Application Number | 20140243227 14/124884 |
Document ID | / |
Family ID | 44511945 |
Filed Date | 2014-08-28 |
United States Patent
Application |
20140243227 |
Kind Code |
A1 |
Clevers; Johannes C. ; et
al. |
August 28, 2014 |
CULTURE MEDIA FOR STEM CELLS
Abstract
Culture media and methods for expanding and differentiating
populations of stem cells and for obtaining organoids. Expanded
cell populations and organoids obtainable by methods of the
invention and their use in drug screening, toxicity assays and
regenerative medicine.
Inventors: |
Clevers; Johannes C.; (Huis
Ter Heide, NL) ; Sato; Toshiro; (Hilversum, NL)
; Huch Ortega; Meritxell; (Utrecht, NL) ; Richard;
Wouter; (Karthaus, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Clevers; Johannes C.
Sato; Toshiro
Huch Ortega; Meritxell
Richard; Wouter |
Huis Ter Heide
Hilversum
Utrecht
Karthaus |
|
NL
NL
NL
NL |
|
|
Assignee: |
Koninklijke Nederlandse Akademie
van Wetenschappen
Utrecht
NL
|
Family ID: |
44511945 |
Appl. No.: |
14/124884 |
Filed: |
June 11, 2012 |
PCT Filed: |
June 11, 2012 |
PCT NO: |
PCT/IB2012/052950 |
371 Date: |
May 9, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13194866 |
Jul 29, 2011 |
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14124884 |
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61594295 |
Feb 2, 2012 |
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61513461 |
Jul 29, 2011 |
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61520569 |
Jun 10, 2011 |
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61571663 |
Jun 30, 2011 |
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Current U.S.
Class: |
506/9 ;
435/283.1; 435/29; 435/32; 435/377; 435/6.12; 506/10 |
Current CPC
Class: |
A61P 3/10 20180101; G01N
33/5008 20130101; C12N 2501/392 20130101; C12N 5/067 20130101; C12N
5/0683 20130101; C12N 5/0688 20130101; C12N 5/0676 20130101; C12N
2501/727 20130101; A61P 1/16 20180101; C12N 2501/119 20130101; C12N
2501/345 20130101; A61P 1/04 20180101; A61P 13/08 20180101; C12N
2501/16 20130101; C12N 5/0679 20130101; C12N 5/0693 20130101; C12N
2500/38 20130101; C12N 2501/02 20130101; C12N 2501/415 20130101;
C12N 5/0018 20130101; C12N 2501/15 20130101; C12N 2501/12
20130101 |
Class at
Publication: |
506/9 ; 435/377;
435/283.1; 435/29; 435/32; 506/10; 435/6.12 |
International
Class: |
C12N 5/071 20060101
C12N005/071; C12N 5/09 20060101 C12N005/09; G01N 33/50 20060101
G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 10, 2011 |
GB |
1111244.8 |
Claims
1. A culture medium for expanding or differentiating a population
of adult stem cells, wherein said culture medium comprises: i. any
one of Rspondin 1-4 and/or an Rspondin mimic; and ii. one or more
TGF-beta inhibitor, wherein the inhibitor is a TGF-beta inhibitor
if it can inhibit TGF-beta signalling in a cellular assay in which
cells are stably transfected with a reporter construct comprising
the human PAI-1 promoter.
2. The culture medium of claim 1, wherein the one or more inhibitor
binds to and reduces the activity of one or more serine/threonine
protein kinases selected from the group consisting of ALK5, ALK4
and ALK7.
3. The culture medium of claim 1, wherein the one or more inhibitor
that directly or indirectly negatively regulates TGF-beta
signalling is selected from the group consisting of A83-01,
SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-2511.
4-6. (canceled)
7. The culture medium of claim 1, wherein the inhibitor is added at
a concentration of between 1 nM and 100 .mu.M, between 10 nM and
100 .mu.M, between 100 nM and 10 .mu.M, or approximately 1 .mu.M,
wherein the total concentration of the one or more inhibitor is
between 10 nM and 100 .mu.M, between 100 nM and 10 .mu.M, or
approximately 1 .mu.M.
8. The culture medium of claim 1, wherein the culture medium
comprises one or more additional components selected from: a BMP
inhibitor, a Wnt agonist, a receptor tyrosine kinase ligand,
nicotinamide, a p38 inhibitor, a Rock inhibitor, gastrin, an
activator of the prostaglandin signalling pathway and
testosterone.
9-28. (canceled)
29. A composition comprising a culture medium according to claim 1
and an extracellular matrix or a 3D matrix that mimics the
extracellular matrix by its interaction with the cellular membrane
proteins such as integrins, for example, a laminin-containing
extracellular matrix such as Matrigel.TM. (BD Biosciences).
30. A hermetically-sealed vessel containing a culture medium or
composition according to claim 1.
31. Use of a culture medium according to claim 1 for expanding or
differentiating a stem cell, population of stem cells, tissue
fragment or organoid.
32-34. (canceled)
35. A method for expanding a single stem cell, a population of stem
cells or a tissue fragment, preferably to obtain an organoid,
wherein the method comprises culturing the single stem cell or
population of stem cells in a culture medium according to claim
1.
36. A method according to claim 35, wherein the method comprises:
providing a stem cell, a population of stem cells or an isolated
tissue fragment; providing a culture medium wherein said culture
medium comprises: i. any one of Rspondin 1-4 and/or an Rspondin
mimic; and ii. one or more TGF-beta inhibitor, wherein the
inhibitor is a TGF-beta inhibitor if it can inhibit TGF-beta
signalling in a cellular assay in which cells are stably
transfected with a reporter construct comprising the human PAI-1
promoter; contacting the stem cells with the culture medium; and
culturing the cells under appropriate conditions.
37. A method according to claim 35, wherein the method comprises
bringing the stem cell, the population of stem cells or the
isolated tissue fragment and the culture medium into contact with
an extracellular matrix or a 3D matrix that mimics the
extracellular matrix by its interaction with the cellular membrane
proteins such as integrins, for example a laminin-containing
extracellular matrix such as Matrigel.TM. (BD Biosciences).
38. (canceled)
39. A method according to claim 35, wherein the method comprises:
culturing the stem cell, population of stem cells or tissue
fragments in a first expansion medium; and continuing to culture
the stem cell, population of stem cells or tissue fragments and
replenishing the medium with a differentiation medium, wherein the
differentiation medium does not comprise one or more of, preferably
all of the factors selected from: a TGF-beta inhibitor, a p38
inhibitor, nicotinamide and Wnt.
40-45. (canceled)
46. The method of claim 35, wherein a Rock inhibitor is added to
the culture medium for the first 1, 2, 3, 4, 5, 6 or 7 days,
optionally every second day.
47. (canceled)
48. An organoid or population of cells obtainable by the method of
claim 35.
49. (canceled)
50. An organoid or population of cells according to claim 48,
wherein the organoid or population of cells has been cultured for
at least 3 months, for example at least 4 months, at least 5
months, at least 6 months, at least 7 months, at least 9 months, or
at least 12 months or more.
51. An organoid or population of cells according to claim 48,
wherein the organoid or population of cells expands at a rate of at
least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at
least 7 fold, at least 8 fold, at least 9 fold or at least 10 fold
per week.
52-53. (canceled)
54. An organoid or population of cells according to claim 48 which
is frozen and stored at below -5.degree. C., below -10.degree. C.,
below -20.degree. C., below -40.degree. C., below -60.degree. C.,
below -80.degree. C., below -100.degree. C., or below -150.degree.
C., for example at approximately -180.degree. C.
55-59. (canceled)
60. A composition comprising: i) one or more organoids or
population of cells according to claim 48; and ii) a culture medium
and/or an extracellular matrix, wherein said culture medium
comprises: i. any one of Rspondin 1-4 and/or an Rspondin mimic; and
ii. one or more TGF-beta inhibitor, wherein the inhibitor is a
TGF-beta inhibitor if it can inhibit TGF-beta signalling in a
cellular assay in which cells are stably transfected with a
reporter construct comprising the human PAI-1 promoter.
61. An organoid according to claim 48 for use in drug screening,
target validation, target discovery, toxicology, toxicology
screens, personalized medicine, regenerative medicine or ex vivo
cell/organ models, for example for use as a disease model.
62. The organoid according to claim 61, wherein the regenerative
medicine or personalized medicine comprises transplantation of said
organoid into a mammal, preferably into a human.
63. A method for screening for a therapeutic or prophylactic drug
or cosmetic, wherein the method comprises: culturing an organoid or
population of cells according to claim 48, with a culture medium,
wherein said culture medium comprises: i. any one of Rspondin 1-4
and/or an Rspondin mimic; and ii. one or more TGF-beta inhibitor,
wherein the inhibitor is a TGF-beta inhibitor if it can inhibit
TGF-beta signalling in a cellular assay in which cells are stably
transfected with a reporter construct comprising the human PAI-1
promoter; exposing said organoid or population of cells to one or a
library of candidate molecules; evaluating said organoid or
population of cells for any effects, for example any change in a
cell, such as a reduction in or loss of proliferation, a
morphological change and/or cell death; and identifying the
candidate molecule that causes said effects as a potential drug or
cosmetic.
Description
[0001] All documents cited herein are incorporated by reference in
their entirety.
TECHNICAL FIELD
[0002] The invention is in the field of stem cell culture media and
methods, in particular culture media and methods for expanding
populations of stem cells, e.g. human epithelial stem cells.
BACKGROUND
[0003] There is great interest in culture media and methods for
expanding populations of stem cells. Populations of stem cells have
many uses. For example, stem cells and their differentiated progeny
can be used in cellular assays, drug screening, and toxicity
assays. Stem cells also show promise for cell-based therapies, such
as in regenerative medicine for the treatment of damaged tissue.
They can also act as a source of differentiated cells for
transplantation purposes e.g. transplantation of pancreatic
beta-cells for treatment of diabetes etc. Furthermore, efficient
cell culture media are important for providing and maintaining
populations of cells for research purposes.
[0004] There is also interest in culture media and methods for
culturing stem cells for the formation, maintenance and expansion
of organoids, such as intestinal crypt-villus, gastric or
pancreatic organoids. An organoid comprises stem cells, such as
epithelial stem cells, which retain their undifferentiated
phenotype and self-renewal properties but also have differentiating
progeny that grow into tissue-like structures. Similarly to
populations of related or identical cells, crypt-villus, gastric or
pancreatic organoids, which more closely mimic the basic physiology
of their tissue of origin, may be used in toxicity assays, or
assays for drugs or food supplements. They may also be useful for
culturing pathogens which currently lack suitable tissue culture or
animal models. Furthermore, such organoids may be useful in
regenerative medicine, for example in post-radiation and/or
post-surgery repair of the intestinal epithelium, or in the repair
of the intestinal epithelium in patients suffering from
inflammatory bowel disease.
[0005] It is clear that there are many clinical and research
applications for stem cells and their differentiated progeny. For
all these applications, reproducible stem cell culture methods are
of the utmost importance for providing adequate numbers of cells of
suitable quality. For example, for effective drug screening,
conditions must be carefully controlled requiring precise culture
methods for controlling differentiation and proliferation of cells,
so that pure populations of phenotypically and karyotypically
identical cells can be generated. Similarly, for cell-based
therapies, wherein cultured cells may be directly provided to
patients, the cells must be genetically and phenotypically sound so
as to avoid undesirable immune responses or cell fates when
provided to the patient.
[0006] Although a variety of culture systems have been described
for culturing primary epithelial stem cells, including intestinal
epithelial stem cells (Bjerknes and Cheng, 2006. Methods Enzymol.
419: 337-83), to date, no long-term culture system has been
established which maintains the differentiation potential and
phenotypic and genomic integrity of human epithelial stem
cells.
[0007] International patent application WO2010/090513 discloses a
method for culturing epithelial stem cells or isolated tissue
fragments. The method is optimised for the culturing of human colon
and intestinal crypts by the addition of Wnt-3a to the medium. This
was the first time that human intestinal stem cell cultures had
been cultured for a prolonged period of time (up to 3 months) and
provided the first reproducible human intestinal stem cell culture
system. However, there is still a need for improved stem cell
culture media and methods, in particular human stem cell culture
media and methods, that improve proliferation rates, survival time
and phenotypic and genomic integrity of stem cells grown in
culture.
SUMMARY OF THE INVENTION
[0008] The invention provides improved culture media and methods
for stem cells, in particular human epithelial stem cells, and
organoids comprising said stem cells, which provide significant
advantages over known culture media and methods. The invention also
provides related culture medium supplements, compositions and
uses.
[0009] Accordingly, the invention provides a culture medium for
expanding a population of stem cells, wherein the culture medium
comprises at least one or more inhibitors that bind to and reduce
the activity of one or more serine/threonine protein kinase
targets. This has the effect of allowing continual growth for at
least 3 months at an expansion rate of approximately five-fold
expansion per week. The serine/threonine protein kinase is
preferably selected from the group comprising: TGFbeta receptor
kinase 1, ALK4, ALK5, ALK7, p38. Surprisingly, the inventors have
found that the inclusion of inhibitors of certain serine/threonine
kinases in culture media significantly improved the performance of
the culture media in expanding a population of stem cells. The
population of stem cells may be normal (healthy) cells or diseased
cells (for example, cancer stem cells). Specifically, inhibitors of
p38 and ALK were shown to provide the greatest improvement out of
all the compounds tested. This is unexpected because there is no
known mechanism predicting how these particular inhibitors might
work. Indeed, several of the small molecule inhibitors that were
chosen to be tested and function in similar pathways, had no effect
on the method. Therefore, the skilled person could not have
predicted that inhibitors of these particular kinases would have
such a marked improvement on the culture medium. A still further
improvement was observed when two inhibitors, for example a p38
inhibitor, such as SB202190 and an ALK inhibitor, such as A83-01,
were added to the culture medium together.
[0010] To arrive at this realisation, the inventors investigated
signalling pathways that are known to be subverted in certain
cancers e.g. colorectal cancer. They hypothesised that these
pathways, which affect cell fate in cancer, may also play a role in
determining cell fate in culture conditions. It should be
emphasised, however, that this hypothesis was entirely new; given
the state of the art, there was no way to predict the effect of any
of these additional compounds on the culture medium, and no
particular expectation that any of these compounds might in fact
have a beneficial effect.
[0011] In a first screening experiment, a series of vitamins,
hormones and growth factors were tested in combination with
standard stem cell culture media. Gastrin and nicotinamide were
initially identified as resulting in significantly improved culture
conditions. Incorporating these factors into the standard culture
conditions, a second screening experiment was performed, in which
small molecule inhibitors related to relevant signalling pathways,
such as ERK, p38, JNK, PTEN, ROCK, and Hedgehog, were tested. These
pathways were chosen because they were known to be subverted in
certain cancers.
[0012] Previous attempts to culture human intestinal stem cells
with previously described stem cell culture medium (comprising
Epidermal Growth Factor (EGF or ("E"), Noggin ("N") and R-spondin
("R"), referred to herein as "ENR" medium) optimised with Wnt-3A
("W") (referred to herein as "WENR" medium), have resulted in the
disintegration of most cells within 7 days, with very few cells
surviving beyond 1 month. Such attempts have also been subject to
slow proliferation times, chromosome irregularities and
morphological changes from budding to cystic structures. By
"cystic" it is meant that the organoid is mostly spherical. By
"budding" it is meant that the organoid has multiple regions
growing out of the basic structure. It is not necessarily always an
advantage to have budding structures, although budding structures
typically have a larger surface area and typically resemble the
corresponding in vivo tissue more closely.
[0013] The inventors showed that the improved method allowed
continual growth of the stem cells for at least seven months.
[0014] The new method also increased the speed of proliferation of
the cells in the expanded population. This is clearly of great
utility when growing cells for commercial and therapeutic
purposes.
[0015] The new method also increased the quality of the cells in
the expanded population. This is a great advantage because clinical
and research applications for stem cells and their differentiated
progeny require reproducible stem cell culture methods that provide
populations of cells of high quality. Generally, in vitro expansion
of stem cells aims to provide a population of cells which resemble
their in vivo counterparts as closely as possible. This property is
herein referred to as the "genomic and phenotypic integrity" of the
cells.
[0016] For the first time, the inventors have discovered that it is
possible to expand human epithelial stem cells in culture, without
loss of genomic and phenotypic integrity, for at least 7 months
(see Example 1). Under the improved culture conditions of the
invention, human intestinal organoids displayed budding organoid
structures, rather than the cystic structures seen under previous
culture conditions. Metaphase spreads of organoids more than 3
months old consistently revealed 46 chromosomes in each of the 20
cells taken from three different donors. Furthermore, microarray
analysis revealed that the stem cells in culture possessed similar
molecular signatures to intestinal crypt cells including intestinal
stem cell genes.
[0017] The inventors also demonstrated that the human intestinal
organoids generated by media and methods of the present invention,
mimicked in vivo cell fate decisions in response to external
factors. For example, it has previously been shown that Notch
inhibition in intestinal stem cells, terminates intestinal
epithelial proliferation and induces goblet cell hyperplasia in
vivo. The inventors were able to show that the intestinal organoids
of the invention, when treated with a Notch inhibitor, ceased
proliferation and most cells converted into goblet cells within 3
days.
[0018] Similar advantages were observed when including a TGF-beta
inhibitor and/or a p38 inhibitor in culture media for expanding
stem cells or organoids from other epithelial tissues, such as
stomach, pancreas, liver and prostate (see the Examples). The
tissues may be normal (healthy) tissues or diseased tissues, for
example cancerous tissues or tissues showing a cystic fibrosis
phenotype.
[0019] These results show the dramatic improvement in the genomic
and phenotypic integrity of the stem cells and organoids produced
by the methods and media of the present invention compared to
previous methods and media.
[0020] Thus, the invention provides a culture medium for expanding
and/or differentiating a population of adult stem cells, wherein
said culture medium comprises: [0021] i. any one of Rspondin 1-4
and/or an Rspondin mimic; and [0022] ii. one or more inhibitor that
directly or indirectly negatively regulates TGF-beta
signalling.
[0023] The invention also provides a composition comprising a
culture medium according to the invention and an extracellular
matrix or a 3D matrix that mimics the extracellular matrix by its
interaction with the cellular membrane proteins such as integrins,
for example, a laminin-containing extracellular matrix such as
Matrigel.TM. (BD Biosciences).
[0024] The invention also provides a hermetically-sealed vessel
containing a culture medium or composition according to the
invention.
[0025] The invention also provides the use of a culture medium
according to the invention for expanding and/or differentiating a
stem cell, population of stem cells, tissue fragment or
organoid.
[0026] The invention also provides methods for expanding a single
stem cell, a population of stem cells or a tissue fragment,
preferably to generate an organoid, wherein the method comprises
culturing the single stem cell or population of stem cells in a
culture medium according to the invention.
[0027] The invention also provides organoids or populations of
cells obtainable by the methods of the invention.
[0028] The invention also provides an organoid, preferably
obtainable by the methods of the invention, which is a
three-dimensional organoid comprising epithelial cells surrounding
a central lumen, wherein optionally the epithelial cells exist in
distinct dividing domains and differentiating domains.
[0029] The invention also provides an organoid, preferably
obtainable by the methods of the invention, which is a
three-dimensional organoid comprising epithelial cells arranged in
regions of monolayers, optionally folded monolayers and regions of
stratified cells, and preferably which is a three-dimensional
organoid comprising epithelial cells surrounding a central lumen,
wherein optionally the epithelial cells exist in distinct dividing
domains and differentiating domains.
[0030] The invention also provides a composition comprising: [0031]
i) one or more organoids or population of cells of the invention;
and [0032] ii) a culture medium of the invention and/or an
extracellular matrix.
[0033] The invention also provides an organoid, a population of
cells or a composition according to the invention for use in drug
screening, target validation, target discovery, toxicology,
toxicology screens, personalized medicine, regenerative medicine or
ex vivo cell/organ models, for example for use as a disease
model.
[0034] The invention also provides an organoid, a population of
cells or a composition according to the invention, for use in
transplantation of said organoid, population of cells or
composition into a mammal, preferably into a human.
[0035] The invention also provides a population of stem cells, or
organoids comprising said stem cells, that have been obtained or
are obtainable using the culture medium of the invention. The stem
cells or organoids comprising said stem cells may be used, for
example, for transplantation purposes or other therapeutic
applications. For example, the stem cells or organoids comprising
said stem cells may be used for drug screening, target validation,
target discovery, toxicology and toxicology screens, personalized
medicine, regenerative medicine and ex vivo cell/organ models, for
example disease models.
[0036] The invention also provides compositions comprising a
culture medium of the invention.
[0037] The invention also provides culture medium supplements
comprising an inhibitor according to the invention.
[0038] The invention also provides a hermetically-sealed vessel
comprising a culture medium and/or a culture medium supplement
according to the invention.
[0039] The specific ingredients of the culture media, supplements
and compositions of the invention can be varied according to
particular needs and applications. Likewise, the precise steps of
the methods of the invention can vary according to particular needs
and applications.
[0040] The culture media, supplements, methods, compositions and
uses according to this invention may also be optimised by routine
experimentation. For example, if a culture medium, supplement or
composition fails to give the desired level of stem cell expansion,
variables such as the amount of each ingredient in the culture
medium or supplement, seeding densities, culture conditions,
culture periods, etc. can be altered in further experiments. The
amount of each of the ingredients described herein can be optimised
independently of the other ingredients by routine optimisation or
one or more ingredients can be added or removed. A culture medium
can be tested for its ability to support expansion of stem cells by
testing it alongside or in place of a known culture medium or
method.
[0041] The culture media, supplements, methods, compositions and
uses of the invention are described in more detail below. The
practice of the present invention will employ, unless otherwise
indicated, conventional techniques of cell culture, molecular
biology and microbiology, which are within the skill of those
working in the art.
[0042] Numerous textbooks are available that provide guidance on
mammalian cell culture media and methods, including textbooks
dedicated to culture media and methods for culturing stem cells.
Such textbooks include `Basic Cell Culture Protocols` by J. Pollard
and J. M. Walker (1997), `Mammalian Cell Culture: Essential
Techniques` by A. Doyle and J. B. Griffiths (1997), `Culture of
Animal Cells: A Manual of Basic Technique` by R. I. Freshney
(2005), `Basic Cell Culture Protocols` by C. Helgason and C. L.
Miller (2005), `Stem Cells: From Bench to Bedside` by A. Bongso
(2005), `Human Stem Cell Manual: A Laboratory Guide` by J. F.
Loring, R. L. Wesselschmidt and P. H. Schwartz (2007).
[0043] Stem cells and cell culture reagents and apparatus for use
in the invention are available commercially, e.g. from Cellartis AB
(Goteborg, Sweden), VitroLife AB (Kungsbacka, Sweden), GIBCO.RTM.
(Invitrogen), Millipore Corporation (Billerica, Mass.), Sigma.RTM.
(St. Louis, Mo.) and Biomol International L.P. (Exeter, UK).
DETAILED DESCRIPTION
[0044] According to the invention, there is provided a culture
medium for expanding a population of stem cells, wherein the
culture medium comprises at least one or more inhibitors that bind
to and reduce the activity of one or more serine/threonine protein
kinase targets, wherein the culture medium has the effect of
allowing continual growth of the population of stem cells for at
least 3 months, preferably at least 4 months, at least 5 months, at
least 6 months, at least 7 months, at least 9 months, or at least
12 months or more.
Inhibitors
[0045] A culture medium used according to a first aspect of the
invention comprises any inhibitor that, directly or indirectly,
negatively regulates TGF-beta or p38 signalling. In a preferred
embodiment the culture medium of the invention comprises an
inhibitor that directly or indirectly negatively regulates TGF-beta
signalling. In some embodiments the culture medium of the invention
comprises an inhibitor that directly or indirectly negatively
regulates TGF-beta and an inhibitor that directly or indirectly
negatively regulates p38 signalling. In a further embodiment, the
culture medium of the invention additionally comprises Rspondin or
an Rspondin mimic.
[0046] The one or more inhibitor preferably targets a
serine/threonine protein kinase selected from the group comprising:
TGF-beta receptor kinase 1, ALK4, ALK5, ALK7, p38. An inhibitor of
any one of these kinases is one that effects a reduction in the
enzymatic activity of any one (or more) of these molecules
Inhibition of ALK and p38 kinase has previously been shown to be
linked in B-cell lymphoma (Bakkebo M Huse K, Hilden V I, Smeland E
B, Oksvold M P, "TGF-beta-induced growth inhibition in B-cell
lymphoma correlates with Smad1/5 signalling and constitutively
active p38 MAPK", BMC Immunol 11:57, 2010). In this publication, it
was found that TGF-beta sensitive cell lines expressed higher cell
surface levels of ALK-5 and that constitutive phosphorylation of
p38 was restricted to the TGF-beta sensitive cell lines Inhibition
of p38 MAPK led to reduced sensitivity to TGF-beta suggesting that
phosphorylation of Smad1/5 is important for the anti-proliferative
effects of TGF-beta in B-cell lymphoma. The results indicate a role
for p38 MAPK in the regulation of TGF-beta-induced
anti-proliferative effects.
[0047] Without wishing to be bound by theory, the present inventors
propose that ALK and p38 belong to a pathway that negatively
regulates long-term maintenance of stem cells, in particular, human
epithelial stem cells. The inventors hypothesise that inhibitors
that act at any level on this pathway, including, for example, by
inhibiting Smad1/5 signalling, would also be beneficial for stem
cell culture. Smads play a key role in TGF-beta signalling.
[0048] In some embodiments an inhibitor of the invention binds to
and reduces the activity serine/threonine protein kinase selected
from the group comprising: TGF-beta receptor kinase 1, ALK4, ALK5,
ALK7, p38.
[0049] In some embodiments of the invention, the culture medium
comprises a TGF-beta inhibitor, meaning any inhibitor that,
directly or indirectly, negatively regulates TGF-beta signalling.
In some embodiments, a culture medium of the invention comprises
one or more TGF-beta inhibitor that binds to and reduces the
activity of one or more serine/threonine protein kinases selected
from the group consisting of ALK5, ALK4, TGF-beta receptor kinase 1
and ALK7.
[0050] ALK4, ALK5 and ALK7 are all closely related receptors of the
TGF-beta superfamily. ALK4 has GI number 91; ALK5 (also known as
TGF-beta receptor kinase 1) has GI number 7046; and ALK7 has GI
number 658. In one embodiment, an inhibitor according to the
invention binds to and reduces the activity of ALK4, ALK5 (TGF-beta
receptor kinase 1) and/or ALK7. In another embodiment, the TGF-beta
receptor binds to and reduces the activity of a Smad protein, for
example R-SMAD or SMAD1-5 (i.e. SMAD 1, SMAD 2, SMAD 3, SMAD 4 or
SMAD 5). In a preferred embodiment, the culture medium of the
invention comprises an inhibitor of ALK5.
[0051] Various methods for determining if a substance is a TGF-beta
inhibitor are known. For example, a cellular assay may be used, in
which cells are stably transfected with a reporter construct
comprising the human PAI-1 promoter or Smad binding sites, driving
a luciferase reporter gene. Inhibition of luciferase activity
relative to control groups can be used as a measure of compound
activity (De Gouville et al., Br J Pharmacol. 2005 May; 145(2):
166-177). Another example is the AlphaScreen.RTM. phosphosensor
assay for measurement of kinase activity (Drew A E et al.,
Comparison of 2 Cell-Based Phosphoprotein Assays to Support
Screening and Development of an ALK Inhibitor J Biomol Screen.
16(2) 164-173, 2011).
[0052] Various TGF-beta inhibitors are known in the art (for
example, see Table 1). In some embodiments the inhibitor that
directly or indirectly negatively regulates TGF-beta signalling is
selected from the group consisting of A83-01, SB-431542, SB-505124,
SB-525334, SD-208, LY-36494 and SJN-2511.
[0053] In some embodiments of the invention, the culture medium
comprises a p38 inhibitor, meaning any inhibitor that, directly or
indirectly, negatively regulates p38 signalling. In some
embodiments, an inhibitor according to the invention binds to and
reduces the activity of p38 (GI number 1432). p38 protein kinases
are part of the family of mitogen-activated protein kinases
(MAPKs). MAPKs are serine/threonine-specific protein kinases that
respond to extracellular stimuli, such as environmental stress and
inflammatory cytokines, and regulate various cellular activities,
such as gene expression, mitosis, differentiation, proliferation,
and cell survival/apoptosis. The p38 MAPKs exist as a, .beta.,
.beta.2, .gamma. and .delta. isoforms. A p38 inhibitor is an agent
that binds to and reduces the activity of at least one p38 isoform.
Various methods for determining if a substance is a p38 inhibitor
are known, and might be used in conjunction with the invention.
Examples include: phospho-specific antibody detection of
phosphorylation at Thr180/Tyr182, which provides a well-established
measure of cellular p38 activation or inhibition; biochemical
recombinant kinase assays; tumor necrosis factor alpha (TNF.alpha.)
secretion assays; and DiscoverRx high throughput screening platform
for p38 inhbitors (see
http://www.discoverx.com/kinases/literature/biochemical/collaterals/DRx_p-
oster_p38%20KBA. pdf). Several p38 activity assay kits also exist
(e.g. Millipore, Sigma-Aldrich).
[0054] The inventors hypothesise that in some embodiments, high
concentrations (e.g. more than 100 nM, or more than 1 uM, more than
10 uM, or more than 100 uM) of a p38 inhibitor may have the effect
of inhibiting TGF-beta. However, the inventors do not wish to be
constrained by this hypothethis and in other embodiments, the p38
inhibitor does not inhibit TGF-beta signalling.
[0055] Various p38 inhibitors are known in the art (for example,
see Table 1). In some embodiments, the inhibitor that directly or
indirectly negatively regulates p38 signalling is selected from the
group consisting of SB-202190, SB-203580, VX-702, VX-745,
PD-169316, RO-4402257 and BIRB-796. In a further embodiment of the
invention, the culture medium comprises both: a) an inhibitor that
binds to and reduces the activity of any one or more of the kinases
from the group consisting of: ALK4, ALK5 and ALK7; and b) an
inhibitor that binds to and reduces the activity of p38. In a
preferred embodiment, the culture medium comprises an inhibitor
that binds to and reduces the activity of ALK5 and an inhibitor
that binds to and reduces the activity of p38.
[0056] In one embodiment, an inhibitor according to the invention
binds to and reduces the activity of its target (for example,
TGF-beta or p38) by more than 10%; more than 30%; more than 60%;
more than 80%; more than 90%; more than 95%; or more than 99%
compared to a control, as assessed by a cellular assay. Examples of
cellular assays for measuring target inhibition are well known in
the art as described above.
[0057] An inhibitor according to the invention may have an IC50
value equal to or less than 2000 nM; less than 1000 nM; less than
100 nM; less than 50 nM; less than 30 nM; less than 20 nM or less
than 10 nM. The IC50 value refers to the effectiveness of an
inhibitor in inhibiting its target's biological or biochemical
function. The IC50 indicates how much of a particular inhibitor is
required to inhibit a kinase by 50%. IC50 values can be calculated
in accordance with the assay methods set out above.
[0058] An inhibitor according to the invention may act
competitively, non-competitively, uncompetitively or by mixed
inhibition. For example, in certain embodiments, an inhibitor may
be a competitive inhibitor of the ATP binding pocket of the target
kinase.
[0059] Inhibitors according to the invention may exist in various
forms, including natural or modified substrates, enzymes,
receptors, small organic molecules, such as small natural or
synthetic organic molecules of up to 2000 Da, preferably 800 Da or
less, peptidomimetics, inorganic molecules, peptides, polypeptides,
antisense oligonucleotides aptamers, and structural or functional
mimetics of these including small molecules. The inhibitor
according to the invention may also be an aptamer. As used herein,
the term "aptamer" refers to strands of oligonucleotides (DNA or
RNA) that can adopt highly specific three-dimensional
conformations. Aptamers are designed to have high binding
affinities and specificities towards certain target molecules,
including extracellular and intracellular proteins.
[0060] For example, the inhibitor may be a small synthetic molecule
with a molecular weight of between 50 and 800 Da, between 80 and
700 Da, between 100 and 600 Da or between 150 and 500 Da.
[0061] In some embodiments, the small-molecule inhibitor comprises
a pyridinylimidazole or a 2,4 disubstituted pteridine or a
quinazoline, for example comprises:
##STR00001##
[0062] Particular examples of inhibitors that may be used in
accordance with the invention include, but are not limited to:
SB-202190, SB-203580, SB-206718, SB-227931, VX-702, VX-745,
PD-169316, RO-4402257, BIRB-796, A83-01 SB-431542, SB-505124,
SB-525334, LY 364947, SD-208, SJN 2511 (see table 1). A culture
medium of the invention may comprise one or more of any of the
inhibitors listed in table 1. A culture medium of the invention may
comprise any combination of one inhibitor with another inhibitor
listed. For example, a culture medium of the invention may comprise
SB-202190 or SB-203580 or A83-01; or a culture medium of the
invention may comprise SB-202190 and A83-01; or a culture medium of
the invention may comprise SB-203580 and A83-01. The skilled person
will appreciate that other inhibitors and combinations of
inhibitors which bind to and reduce the activity of the targets
according to the invention, may be included in a culture medium or
a culture medium supplement in accordance with the invention.
[0063] Inhibitors according to the invention may be added to the
culture medium to a final concentration that is appropriate, taking
into account the IC50 value of the inhibitor.
[0064] For example, SB-202190 may be added to the culture medium at
a concentration of between 50 nM and 100 uM, or between 100 nM and
50 uM, or between 1 uM and 50 uM. For example, SB-202190 may be
added to the culture medium at approximately 10 uM.
[0065] SB-203580 may be added to the culture medium at a
concentration of between 50 nM and 100 uM, or between 100 nM and 50
uM, or between 1 uM and 50 uM. For example, SB-203580 may be added
to the culture medium at approximately 10 uM.
[0066] VX-702 may be added to the culture medium at a concentration
of between 50 nM and 100 uM, or between 100 nM and 50 uM, or
between 1 uM and 25 uM. For example, VX-702 may be added to the
culture medium at approximately 5 uM.
[0067] VX-745 may be added to the culture medium at a concentration
of between 10 nM and 50 uM, or between 50 nM and 50 uM, or between
250 nM and 10 uM. For example, VX-745 may be added to the culture
medium at approximately 1 uM.
[0068] PD-169316 may be added to the culture medium at a
concentration of between 100 nM and 200 uM, or between 200 nM and
100 uM, or between 1 uM and 50 uM. For example, PD-169316 may be
added to the culture medium at approximately 20 uM.
[0069] RO-4402257 may be added to the culture medium at a
concentration of between 10 nM and 50 uM, or between 50 nM and 50
uM, or between 500 nM and 10 uM. For example, RO-4402257 may be
added to the culture medium at approximately 1 uM.
[0070] BIRB-796 may be added to the culture medium at a
concentration of between 10 nM and 50 uM, or between 50 nM and 50
uM, or between 500 nM and 10 uM. For example, BIRB-796 may be added
to the culture medium at approximately 1 uM.
[0071] A83-01 may be added to the culture medium at a concentration
of between 10 nM and 10 uM, or between 20 nM and 5 uM, or between
50 nM and 1 uM. For example, A83-01 may be added to the culture
medium at approximately 500 nM.
[0072] SB-431542 may be added to the culture medium at a
concentration of between 80 nM and 80 uM, or between 100 nM and 40
uM, or between 500 nM and 10 uM. For example, SB-431542 may be
added to the culture medium at approximately 1 uM.
[0073] SB-505124 may be added to the culture medium at a
concentration of between 40 nM and 40 uM, or between 80 nM and 20
uM, or between 200 nM and 1 uM. For example, SB-505124 may be added
to the culture medium at approximately 500 nM.
[0074] SB-525334 may be added to the culture medium at a
concentration of between 10 nM and 10 uM, or between 20 nM and 5
uM, or between 50 nM and 1 uM. For example, SB-525334 may be added
to the culture medium at approximately 100 nM.
[0075] LY 36494 may be added to the culture medium at a
concentration of between 40 nM and 40 uM, or between 80 nM and 20
uM, or between 200 nM and 1 uM. For example, LY 36494 may be added
to the culture medium at approximately 500 nM.
TABLE-US-00001 TABLE 1 Exemplary inhibitors according to the
invention IC50 Inhibitor Targets (nM) Mol Wt Name Formula A83-01
ALK5 12 421.52 3-(6-Methyl-2- C25H19N5S (TGF-.beta.R1)
pyridinyl)-N-phenyl-4- ALK4 45 (4-quinolinyl)-1H- ALK7 7.5
pyrazole-1- carbothioamide SB-431542 ALK5 94 384.39
4-[4-(1,3-benzodioxol- C22H16N4O3 ALK4 5-yl)-5-(2-pyridinyl)- ALK7
1H-imidazol-2- yl]benzamide SB-505124 ALK5 47 335.4
2-(5-benzo[1,3]dioxol- C20H21N3O2 ALK4 129 5-yl-2-tert-butyl-
3Himidazol- 4-yl)-6-methylpyridine hydrochloride hydrate SB-525334
ALK5 14.3 343.42 6-[2-(1,1- C21H21N5 Dimethylethyl)-5-(6-
methyl-2-pyridinyl)- 1H-imidazol-4- yl]quinoxaline SD-208 ALK5 49
352.75 2-(5-Chloro-2- C17H10ClFN6 fluorophenyl)-4-[(4-
pyridyl)amino]pteridine LY-36494 TGR-.beta.RI 59 272.31
4-[3-(2-Pyridinyl)-1H- C17H12N4 TGF-.beta.RII 400
pyrazol-4-yl]-quinoline MLK-7K 1400 LY364947 ALK5 59 272.30
4-[3-(2-pyridinyl)-1H- C.sub.17H.sub.12N.sub.4
pyrazol-4-yl]-quinoline SJN-2511 ALK5 23 287.32 2-(3-(6- C17H13N5
Methylpyridine-2-yl)- 1H-pyrazol-4-yl)-1,5- naphthyridine SB-202190
p38 MAP 38 331.35 4-[4-(4-Fluorophenyl)- C20H14N3OF kinase
5-(4-pyridinyl)-1H- p38.alpha. 50 imidazol-2-yl]phenol p38.beta.
100 SB-203580 p38 50 377.44 4-[5-(4-Fluorophenyl)- C21H16FN3OS
p38.beta.2 500 2-[4- (methylsulfonyl)phenyl]- 1H-imidazol-4-
yl]pyridine VX-702 p38.alpha. 4-20; 404.32 6- C19H12F4N4O2 (Kd =
[(Aminocarbonyl)(2,6- 3.7) difluorophenyl)amino]- p38.beta. Kd = 17
2-(2,4-difluorophenyl)- 3-pyridinecarboxamide VX-745 p38.alpha. 10
436.26 5-(2,6-Dichlorophenyl)- C19H9Cl2F2N3OS 2-[2,4-
difluorophenyl)thio]- 6H-pyrimido[1,6- b]pyridazin-6-one PD-169316
p38 89 360.3 4-[5-(4-fluorophenyl)- C20H13FN4O
2-(4-nitrophenyl)-1H- imidazol-4-yl]-pyridine RO- p38.alpha. 14
Pyrido[2,3-d]pyrimidin- 4402257 p38.beta. 480 7(8H)-one,6-(2,4-
difluorophenoxy)-2-[[3- hydroxy-1-(2- hydroxyethyl)propyl]amino]-
8-methyl- BIRB-796 p38 4 527.67 1-[2-(4-methylphenyl)- C31H37N5O3
5-tert-butyl-pyrazol-3- yl]-3-[4-(2-morpholin-
4-ylethoxy)naphthalen- 1-yl]urea::3-[2-(4- methylphenyl)-5-tert-
butyl-pyrazol-3-yl]-1- [4-(2-morpholin-4- ylethoxy)naphthalen-1-
yl]urea::3-[3-tert-butyl- 1-(4-methylphenyl)-
1H-pyrazol-5-yl]-1-{4- [2-(morpholin-4- yl)ethoxy]naphthalen-
1-yl}urea
[0076] SD-208 may be added to the culture medium at a concentration
of between 40 nM and 40 uM, or between 80 nM and 20 uM, or between
200 nM and 1 uM. For example, SD-208 may be added to the culture
medium at approximately 500 nM.
[0077] LY364947 may be added to the culture medium at a
concentration of between 40 nM and 40 uM, or between 80 nM and 20
uM, or between 200 nM and 1 uM. For example, LY364947 may be added
to the culture medium at approximately 500 nM.
[0078] SJN 2511 may be added to the culture medium at a
concentration of between 20 nM and 20 uM, or between 40 nM and 10
uM, or between 100 nM and 1 uM. For example, SJN 2511 may be added
to the culture medium at approximately 200 nM.
[0079] Thus, in some embodiments the inhibitor that directly or
indirectly, negatively regulates TGF-beta or p38 signalling is
added to the culture medium at a concentration of between 1 nM and
100 .mu.M, between 10 nM and 100 .mu.M, between 100 nM and 10
.mu.M, or approximately 1 .mu.M, for example, wherein the total
concentration of the one or more inhibitor is between 10 nM and 100
.mu.M, between 100 nM and 10 .mu.M, or approximately 1 .mu.M.
[0080] Additionally to the inhibitor, cell culture media generally
contain a number of components which are necessary to support
maintenance and/or expansion of the cultured cells. A cell culture
medium of the invention will therefore normally contain many other
components in addition to an inhibitor according to the invention.
Suitable combinations of components can readily be formulated by
the skilled person, taking into account the following disclosure. A
culture medium according to the invention will generally be a
nutrient solution comprising standard cell culture components, such
as amino acids, vitamins, inorganic salts, a carbon energy source,
and a buffer as described in more detail below. Other standard cell
culture components that may be included in the culture include
hormones, such as progesterone, proteins, such as albumin,
catalase, insulin and transferrin. These other standard cell
culture components make up the "basal" culture medium.
[0081] A culture medium according to the invention may be generated
by modification of an existing cell culture medium. The skilled
person will understand from common general knowledge the types of
culture media that might be used for stem cell culture. Potentially
suitable cell culture media are available commercially, and
include, but are not limited to, Dulbecco's Modified Eagle Media
(DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM),
Glasgow Minimal Essential Medium (G-MEM), Basal Medium Eagle (BME),
DMEM/Ham's F12, Advanced DMEM/Ham's F12, Iscove's Modified
Dulbecco's Media and Minimal Essential Media (MEM), Ham's F-10,
Ham's F-12, Medium 199, and RPMI 1640 Media. Thus, in some
embodiments, one of these pre-existing cell culture media is used
as the basal culture medium to which is added the inhibitor that,
directly or indirectly, negatively regulates TGF-beta or p38
signalling, and, optionally, to which is added one or more other
components as described herein.
[0082] In some embodiments, the culture medium of the invention
comprises one or more additional components selected from: a BMP
inhibitor, a Wnt agonist, a receptor tyrosine kinase ligand, a Rock
inhibitor, nicotinamide and gastrin. In some embodiments, the
culture medium of the invention comprises any one of Rspondin 1-4
and/or an Rspondin mimic, a TGF-beta inhibitor, a BMP inhibitor
(for example, Noggin) and a Wnt agonist (for example, Wnt(3a)).
[0083] In some embodiments, the culture medium of the invention
comprises any one of Rspondin 1-4 and/or an Rspondin mimic, a BMP
inhibitor (for example, Noggin), a TGF-beta inhibitor, a receptor
tyrosine kinase ligand (for example, EGF), Nicotinamide, a Wnt
agonist (for example, Wnt(3a)), and optionally one or more
additional components selected from: a p38 inhibitor, gastrin,
FGF10, HGF and a Rock inhibitor. The optional additional components
may be added for optimisation of the culture medium for culturing
cells originating from particular tissues, as explained in more
detail later on.
[0084] The culture media of the invention may comprise one or more
bone morphogenetic protein (BMP) inhibitor. BMP ligands signal as
dimers by assembling a quadripartite transmembrane serine/threonine
kinase receptor complex consisting of two type I and two type II
receptors. Complex assembly initiates a phosphorylation cascade
activating the BMP responsive Smads1/5/8 and resulting in changes
in transcriptional activity. Advantageously, the present inventors
show that BMP inhibitors promote expression of Lgr5, and so the
presence of a BMP inhibitor in a culture medium of the invention
will likely result in more proliferative organoids than if the BMP
inhibitor is absent (for example, see Example 3). Thus, BMP
inhibitors are an advantageous component of expansion media of the
invention. Thus, the use of a BMP inhibitor is advantageous in the
use of an expansion medium when it is desirable to culture the
cells for at least 3 months (e.g. at least 4, 5, 6, 7, 8 or 9
months) without the cells differentiating.
[0085] Several classes of natural BMP-binding proteins are known,
including Noggin (Peprotech), Chordin and chordin-like proteins
(R&D systems) comprising chordin domains, Follistatin and
follistatin-related protines (R&D systems) comprising a
follistatin domain, DAN and DAN-like proteins (R&D systems)
comprising a DAN cystein-knot domain, sclerostin/SOST (R&D
systems) and apha-2 macroglobulin (R&D systems). A BMP
inhibitor is an agent that binds to a BMP molecule to form a
complex wherein the BMP activity is reduced, for example by
preventing or inhibiting the binding of the BMP molecule to a BMP
receptor. Alternatively, the inhibitor may be an agent that binds
to a BMP receptor and prevents binding of a BMP ligand to the
receptor, for example, an antibody that binds the receptor. A BMP
inhibitor may be a protein or small molecule and may be naturally
occurring, modified, and/or partially or entirely synthetic. A BMP
inhibitor of a culture medium of the invention may be Noggin, DAN,
or DAN-like proteins including Cerberus and Gremlin (R&D
systems). These diffusible proteins are able to bind a BMP ligand
with varying degrees of affinity and inhibit their access to
signalling receptors. A preferred BMP inhibitor for use in a
culture medium of the invention is Noggin. Noggin may be used at
any suitable concentration. In some embodiments, a basal medium of
the culture medium of the invention may comprise between about 10
ng/ml and about 100 ng/ml of Noggin. For example, a culture medium
may comprise at least 10 ng/ml of Noggin, at least 20 ng/ml of
Noggin, at least 50 ng/ml of Noggin, at least 100 ng/ml of Noggin,
approximately 100 ng/ml of Noggin or 100 ng/ml of Noggin. In some
embodiments, a culture medium may comprise less than 200 ng/ml of
Noggin, less than 150 ng/ml of Noggin, less than 100 ng/ml of
Noggin, less than 75 ng/ml of Noggin, less than 50 ng/ml of Noggin
or less than 30 ng/ml of Noggin. The BMP inhibitor may be added to
the culture medium every second day during culturing, or every day
during culturing, or every third day, every fourth day, every fifth
day or as required. BMP inhibitors are particularly advantageous
components of the expansion media, for example for expanding
pancreas, small intestine, colon, liver, prostate stem cells.
However, Noggin has been shown to prevent some differentiation (for
example, see example 3). Therefore, in some embodiments a BMP
inhibitor is excluded from a differentiation medium of the
invention.
[0086] In some embodiments, cells cultured with a BMP inhibitor
have upregulated expression of Lgr5 compared to cells cultured
without a BMP inhibitor. Therefore, addition of a BMP inhibitor
typically results in more proliferative organoids. This is
surprising, because in the literature it is described that BMP
activity is useful for the differentiation of pancreatic cells into
both the ductal (see keratin7 and 19 expression) and endocrine
cells. Thus, the skilled person would expect the inclusion of a BMP
inhibitor, such as Noggin, to decrease proliferation and to
increase differentiation. However, the inventors surprisingly found
that the use of a BMP inhibitor was advantageous because it
resulted in more proliferative organoids and higher expression of
Lgr5. The culture media of the invention may comprise one or more
Wnt agonist. The Wnt signalling pathway is defined by a series of
events that occur when a Wnt protein binds to a cell-surface
receptor of a Frizzled receptor family member. This results in the
activation of Dishevelled family proteins which inhibit a complex
of proteins that includes axin, GSK-3, and the protein APC to
degrade intracellular beta-catenin. The resulting enriched nuclear
beta-catenin enhances transcription by TCF/LEF family transcription
factors. A Wnt agonist is defined as an agent that activates
TCF/LEF-mediated transcription in a cell. Wnt agonists are
therefore selected from true Wnt agonists that bind and activate a
Frizzled receptor family member including any and all of the Wnt
family proteins, an inhibitor of intracellular beta-catenin
degradation, and activators of TCF/LEF. Said Wnt agonist stimulates
a Wnt activity in a cell by at least 10%, more preferred at least
20%, more preferred at least 30%, more preferred at least 50%, more
preferred at least 70%, more preferred at least 90%, more preferred
at least 100%, relative to a level of said Wnt activity in the
absence of said molecule. As is known to a skilled person, a Wnt
activity can be determined by measuring the transcriptional
activity of Wnt, for example by pTOPFLASH and pFOPFLASH Tcf
luciferase reporter constructs (Korinek et al, 1997 Science 275
1784-1787).
[0087] In some embodiments, a Wnt agonist comprises a secreted
glycoprotein including Wnt-1/Int-1, Wnt-2/Irp (InM-related
Protein), Wnt-2b/13, Wnt-3/Int-4, Wnt-3a (R&D sytems), Wnt-4,
Wnt-5a, Wnt-5b, Wnt-6 (Kirikoshi H et al 2001 Biochem Biophys Res
Com 283 798-805), Writ-7a (R&D systems), Wnt-7b, Wnt-8a/8d,
Wnt-8b, Wnt-9a/14, Wnt-9b/14b/15, Wnt-10a, Wnt-10b/12, WnM 1, and
Wnt-16. An overview of human Wnt proteins is provided in "THE WNT
FAMILY OF SECRETED PROTEINS", R&D Systems Catalog, 2004.
Further Wnt agonists include the R-spondin family of secreted
proteins, which is implicated in the activation and regulation of
Wnt signaling pathway and which is comprised of 4 members
(R-spondin 1 (NU206, Nuvelo, San Carlos, Calif.), R-spondin 2
((R&D systems), R-spondin 3, and R-spondin-4), and Norrin (also
called Nome Disease Protein or NDP) (R&D systems), which is a
secreted regulatory protein that functions like a Wnt protein in
that it binds with high affinity to the Frizzled-4 receptor and
induces activation of the Wnt signaling pathway (Kestutis Planutis
et al (2007) BMC Cell Biol 8 12). In some embodiments, one or more
Wnt agonists for use in the invention is an R-spondin mimic, for
example an agonist of Lgr5 such as an anti-Lgr5 antibody. A
small-molecule agonist of the Wnt signaling pathway, an
aminopyrimidine derivative, was recently identified and is also
expressly included as a Wnt agonist (Lm et al (2005) Angew Chem Int
Ed Engl 44, 1987-90).
[0088] In some embodiments, the Wnt agonist is a GSK-inhibitor.
Known GSK-inhibitors comprise small-interfering RNAs (siRNA, Cell
Signaling), lithium (Sigma), kenpaullone (Biomol International,
Leost, M et al (2000) Eur J Biochem 267, 5983-5994),
6-Bromoindirubin-30-acetoxime (Meyer, L et al (2003) Chem Biol 10,
1255-1266), SB 216763 and SB 415286 (Sigma-Aldrich), and
FRAT-family members and FRAT-derived peptides that prevent
interaction of GSK-3 with axin. An overview is provided by Meijer
et al, (2004) Trends in Pharmacological Sciences 25, 471-480, which
is hereby incorporated by reference. Methods and assays for
determining a level of GSK-3 inhibition are known to a skilled
person and comprise, for example, the methods and assay as
described in Liao et al 2004, Endocrinology, 145(6) 2941-2949.
[0089] In some embodiments, the Wnt agonist is an inhibitor of
RNF43 or ZNRF3. The inventors have discovered that RNF43 and ZNRF3
reside in the cell membrane and negatively regulate levels of the
Wnt receptor complex in the membrane, probably by ubiquitination of
Frizzled. Therefore, the inventors hypothesise that inhibition of
RNF43 or ZNRF3 with antagonistic antibodies, RNAi or small molecule
inhibitors would indirectly stimulate the Wnt pathway. RNF43 and
ZNRF3 have a catalytic ring domain (with ubiquitination activity),
which can be targeted in small molecule inhibitor design. Several
anti-RNF43 antibodies and several anti-ZNRF3 antibodies are
available commercially. In some embodiments, such antibodies are
suitable Wnt agonists in the context of the invention.
[0090] In some embodiments, said Wnt agonist is selected from the
group consisting of Wnt-3a, a GSK-inhibitor (such as CHIR99021),
Wnt 5, Wnt-6a, Norrin, and any other Wnt family protein.
[0091] In some embodiments, said Wnt agonist comprises or consists
of any one of Rspondin 1, Rspondin 2, Rspondin 3 or Rspondin 4. In
a preferred embodiment, said Wnt agonist is selected from one or
more of a Wnt family member, R-spondin 1-4, Norrin, and a
GSK-inhibitor. In some embodiments, said Wnt agonist is a GSK-3
inhibitor, such as CHIR99021 (Stemgent 04-0004).
[0092] In some embodiments, CHIR99021 is added to the culture
medium to a final concentration of between 50 nM and 100 uM, for
example between 100 nM and 50 uM, between 1 uM and 10 uM, between 1
uM and 5 uM, or 3 uM. In some embodiments in which a GSK-3
inhibitor is used, the GSK-3 inhibitor is not BIO
(6-bromoindirubin-3'-oxime, Stemgent 04-0003). It was found by the
inventors that the addition of at least one Wnt agonist to the
basal culture medium is essential for proliferation of the
epithelial stem cells or isolated crypts.
[0093] In a further preferred embodiment, said Wnt agonist
comprises or consists of R-spondin 1 or R-spondin-4. R-spondin 1,
R-spondin 2, R-spondin 3 or R-spondin 4 is preferably added to the
basal culture medium at a concentration of at least 50 ng/ml, more
preferred at least 100 ng/ml, more preferred at least 200 ng/ml,
more preferred at least 300 ng/ml, more preferred at least 500
ng/ml. A most preferred concentration of R-spondin 1, R-spondin 2,
R-spondin 3 or R-spondin 4 is approximately 500 ng/ml or 500 ng/ml.
In some embodiments, R-spondin 1, R-spondin 2, R-spondin 3 or
R-spondin 4 is added to the culture medium at a concentration of at
least 500 ng/ml, at least 600 ng/ml, at least 700 ng/ml, at least
800 ng/ml, at least 900 ng/ml, at least 1 ug/ml, at least 1.5 ug/ml
or at least 2 ug/ml. In another preferred embodiment, R-spondin 1,
R-spondin 2,R-spondin 3 or R-spondin 4 is added to the culture
medium at a concentration of approximately 1 ug/ml or 1 ug/ml. In
some embodiments, R-spondin 1, R-spondin 2, R-spondin 3 or
R-spondin 4 is added to the basal culture medium at a concentration
of less than 1000 ng/ml, for example, less than 800 ng/ml, less
than 600 ng/ml, less than 550 ng/ml, less than 500 ng/ml, less than
400 ng/ml, less than 300 ng/ml or less than 200 ng/ml, or less than
100 ng/ml. In some embodiments, two or more (e.g. 2, 3 or 4) of
Rspondin 1, Rspondin 2, Rspondin 3 and Rspondin 4 ("Rspondin 1-4")
are added to the medium. Preferably, when two or more of Rspondin
1-4 are added, the total concentration of Rspondin amounts to the
concentrations described above. Where culture media described
herein are said to comprise "Rspondin 1-4", it is meant that the
medium comprises any one or more of Rspondin 1, Rspondin 2,
Rspondin 3 and Rspondin 4. Where culture media described herein are
said to comprise "Rspondin", it is meant that the medium comprises
any one or more of Rspondin 1, Rspondin 2, Rspondin 3, Rspondin 4
and an Rspondin mimic.
[0094] During culturing of stem cells, said Wnt family member is
preferably added to the culture medium every second day, while the
culture medium is refreshed preferably every fourth day.
[0095] In a preferred embodiment, a Wnt agonist is selected from
the group consisting of R-spondin, Wnt-3a and Wnt-6. More
preferably, R-spondin and Wnt-3a are both used as Wnt agonist. This
combination is particularly preferred since this combination
surprisingly has a synergistic effect on organoid formation.
Preferred concentrations are approximately 500 ng/ml or 500 ng/ml
for R-spondin and approximately 100 ng/ml or 100 ng/ml for
Wnt3a.
[0096] The culture media of the invention may comprise one or more
receptor tyrosine kinase ligands. An example of a receptor tyrosine
kinase ligand for use in the invention is EGF, which is the ligand
for the receptor tyrosine kinase EGFR. Many receptor tyrosine
kinase ligands are also mitogenic growth factors.
[0097] The culture media of the invention may comprise one or more
mitogenic growth factor. The one or more mitogenic growth factor
may be selected from a family of growth factors comprising
epidermal growth factor (EGF, Peprotech), Transforming Growth
Factor-alpha (TGF-alpha, Peprotech), basic Fibroblast Growth Factor
(bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D
Systems), and Keratinocyte Growth Factor (KGF, Peprotech). EGF is a
potent mitogenic factor for a variety of cultured ectodermal and
mesodermal cells and has a profound effect on the differentiation
of specific cells in vivo and in vitro and of some fibroblasts in
cell culture. The EGF precursor exists as a membrane-bound molecule
which is proteolytically cleaved to generate the 53-amino acid
peptide hormone that stimulates cells. A preferred mitogenic growth
factor is EGF. EGF is preferably added to the basal culture medium
at a concentration of between 5 and 500 ng/ml or of at least 5 and
not higher than 500 ng/ml. A preferred concentration is at least
10, 20, 25, 30, 40, 45, or 50 ng/ml and not higher than 500, 450,
400, 350, 300, 250, 200, 150, or 100 ng/ml. A more preferred
concentration is at least 50 and not higher than 100 ng/ml. An even
more preferred concentration is about 50 ng/ml or 50 ng/ml. The
same concentrations could be used for a FGF, preferably for FGF10
or FGF7. If more than one FGF is used, for example FGF7 and FGF10,
the concentration of a FGF is as defined above and refers to the
total concentration of FGF used. During culturing of stem cells,
said mitogenic growth factor is preferably added to the culture
medium every second day, while the culture medium is refreshed
preferably every fourth day. Any member of the FGF family may be
used. Preferably, FGF7 and/or FGF10 is used FGF7 is also known as
KGF (Keratinocyte Growth Factor). In a further preferred
embodiment, a combination of mitogenic growth factors such as, for
example, EGF and KGF, or EGF and BDNF, is added to the basal
culture medium. In a further preferred embodiment, a combination of
mitogenic growth factors such as, for example, EGF and KGF, or EGF
and FGF10, is added to the basal culture medium. The mitogenic
growth factor may be added to a culture media at a concentration of
between 5 and 500 nanogram/ml or at least 5 and not more than 500
nanogram/ml, for example at least 10, 20, 25, 30, 40, 45, or 50
ng/ml and not higher than 500, 450, 400, 350, 300, 250, 200, 150,
or 100 ng/ml. The mitogenic growth factor may be selected from the
group consisting of EGF, TGF-alpha, KGF, FGF7 and FGF. Preferably,
a mitogenic factor is selected from the groups consisting of EGF,
TGF-alpha and KGF or from EGF, TGF-alpha and FGF7 or from EGF,
TGF-alpha and FGF or from EGF and KGF or from EGF and FGF7 or from
EGF and a FGF or from TGF-alpha and KGF or from TGF-alpha and FGF7
or from TGF-alpha and a FGF. EGF may be replaced by TGF-alpha. In
some embodiments, the mitogenic growth factor is hepatocyte growth
factor (HGF). In some embodiments, HGF is added to the culture
medium.
[0098] In some embodiments, the receptor tyrosine kinase ligand is
a mitogenic growth factor, for example selected from a family of
growth factors consisting of epidermal growth factor (EGF),
Transforming Growth Factor-alpha (TGF-alpha), basic Fibroblast
Growth Factor (bFGF), brain-derived neurotrophic factor (BDNF),
Hepatocyte growth factor (HGF) and Keratinocyte Growth Factor
(KGF).
[0099] ROCK inhibitors, such as Y-27632 (10 .mu.M; Sigma), can be
included in any of the media described, in particular in the first
few days of culture before performing cell sorting experiments,
because it is known to avoid anoikis (a form of programmed cell
death which is induced by anchorage-dependent cells detaching from
the surrounding extracellular matrix). Therefore, any of the media
defined herein, may additionally comprise a ROCK inhibitor for the
first few days. In some embodiments, the culture media of the
invention additionally comprises a ROCK inhibitor, such as Y-27632,
for example for the first few days of culture before performing
cell sorting experiments.
[0100] A further embodiment of a method according to the invention
comprises a culture medium comprising a Rock (Rho-kinase)
inhibitor. The addition of a Rock inhibitor was found to prevent
anoikis, especially when culturing single stem cells. Said Rock
inhibitor is preferably selected from
R-(+)-trans-4-(1-aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide
dihydrochloride monohydrate (Y-27632, Sigma-Aldrich),
5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil or HA1077,
Cayman Chemical), and
(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4--
diazepine dihydrochloride (H-1 152, Tocris Bioschience). Said
Rho-kinase inhibitor, for example Y-27632, is preferably added to
the culture medium every second day during the first seven days of
culturing said stem cells. A Rock inhibitor is preferably included
in the medium in the first few days e.g. for the first 1, 2, 3, 4,
5, 6 or 7 days of culture after single cell seeding or after a
split. Any suitable concentration of the Rock inhibitor may be
used, for example, 1-200 uM, 1-100 uM, 5-50 uM or approximately 10
uM. A preferred concentration for Y27632 is 10 uM. Therefore, in
some embodiments, the invention provides a method for culturing
stem cells and/or a method for obtaining an organoid wherein a Rock
inhibitor is added to the culture medium for the first 1, 2, 3, 4,
5, 6 or 7 days, optionally every second day. In some embodiments,
the Rock inhibitor is not added to the culture medium after the
first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
[0101] Addition of a Rock inhibitor is particularly important when
culturing single stem cells (as mentioned above), i.e. when the
starting material for an organoid is a single stem cell. Therefore,
in some embodiments the invention provides a method for obtaining
an organoid, wherein the method comprises culturing stem cells,
optionally single stem cells, wherein a Rock inhibitor is added to
the culture medium for the first 1, 2, 3, 4, 5, 6 or 7 days,
optionally every second day, and optionally not adding the Rock
inhibitor to the culture medium after the first 2, 3, 4, 5, 6, 7,
8, 9 or 10 days.
[0102] The Rock inhibitor is less important, and sometimes not
necessary, when culturing multiple cells, for example when the
starting material for an organoid is a tissue fragment. Therefore,
in some embodiments, the invention provides a method for obtaining
an organoid, wherein the method comprises culturing stem cells,
optionally a tissue fragment, wherein the Rock inhibitor is not
added to the culture medium either at all or after the first 2, 3,
4, 5, 6, 7, 8, 9 or 10 days.
[0103] After the cells are split into multiple cultures, a Rock
inhibitor may be added to the culture medium in the same way,
meaning for the first 1, 2, 3, 4, 5, 6 or 7 days, optionally every
second day, after the split, particularly when the split involves
taking single stem cells from a first culture and placing these
into a second culture. If the split involves taking multiple stem
cells from the first culture and placing these into a second
culture then addition of a Rock inhibitor is less important, and
sometimes not necessary. Therefore, in some embodiments, wherein
the method for obtaining organoids or for culturing stem cells
involves a split, optionally where a single cell is involved in the
split, a Rock inhibitor is added to the new culture medium for the
first 1, 2, 3, 4, 5, 6 or 7 days, optionally every second day,
after the split. In some embodiments, wherein the method for
obtaining organoids or for culturing stem cells involves a split,
optionally where multiple cells are involved in the split, is not
added to the culture medium either at all or after the first 2, 3,
4, 5, 6, 7, 8, 9 or 10 days.
[0104] In yet a further embodiment, a method according to the
invention comprises a culture medium further comprising a Notch
agonist. Notch signaling has been shown to play an important role
in cell-fate determination, as well as in cell survival and
proliferation. Notch receptor proteins can interact with a number
of surface-bound or secreted ligands, including but not limited to
Delta 1, Jagged 1 and 2, and Delta-like 1, Delta-like 3, Delta-like
4. Upon ligand binding, Notch receptors are activated by serial
cleavage events involving members of the ADAM protease family, as
well as an intramembranous cleavage regulated by the gamma
secretase presenilin. The result is a translocation of the
intracellular domain of Notch to the nucleus where it
transcriptionally activates downstream genes. A preferred Notch
agonist is selected from Jagged 1 and Delta 1, or an active
fragment or derivative thereof. A most preferred Notch agonist is
DSL peptide (Dontu et al., 2004. Breast Cancer Res 6. R605-R615)
with the sequence CDDYYYGFGCNKFCRPR. Said DSL peptide is preferably
used at a concentration between 10 .mu.M and 100 nM or at least 10
.mu.M and not higher than 100 nM. The addition of a Notch agonist,
especially during the first week of culturing, increases the
culture efficiency by a factor of 2-3.
[0105] Said Notch agonist is preferably added to the culture medium
every second day during the first seven days of culturing said stem
cells. Therefore, in some embodiments, the invention provides a
method for culturing stem cells and/or a method for obtaining an
organoid wherein a Notch agonist is added to the culture medium for
the first 1, 2, 3, 4, 5, 6 or 7 days, optionally every second day.
In some embodiments, the Notch agonist is not added to the culture
medium after the first 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
[0106] A Notch agonist is defined as a molecule that stimulates a
Notch activity in a cell by at least 10%, more preferred at least
20%, more preferred at least 30%, more preferred at least 50%, more
preferred at least 70%, more preferred at least 90%, more preferred
at least 100%, relative to a level of a Notch activity in the
absence of said molecule. As is known to a skilled person, a Notch
activity can be determined by measuring the transcriptional
activity of Notch, for example by a 4xwtCBF1-luciferase reporter
construct as described (Hsieh et al, 1996 Mol Cell. Biol. 16,
952-959).
[0107] In a further embodiment, the cell culture medium is
supplemented with a gamma-secretase inhibitor, such as DAPT or DBZ
Gamma-secretase inhibitors can influence cell fate decisions during
differentiation. For example, in some embodiments, gamma-secretase
inhibitors can influence cell fate towards secretory cells, such as
goblet cells. Any suitable concentration of the gamma-secretase
inhibitor may be used, for example, between 1 nM and 10 uM, 1 nM
and 1 uM, between 1 and 100 nM, or preferably between 1 and 20 nM.
For example, a gamma-secretase inhibitor may be added to the
culture medium to a final concentration of approximately 1 nM.
[0108] In a further embodiment, the cell culture medium is
supplemented with gastrin (or a suitable alternative such as Leu
15-gastrin). Gastrin (or a suitable alternative) may be added to
the culture medium to a final concentration of between 1 nM and 10
uM, 1 nM and 1 uM, between 5 and 100 nM, or preferably between 10
and 50 nM. For example, Leu15-gastrin may be added to the culture
medium to a final concentration of approximately 10 nM. Gastrin is
not necessary for some culture media of the invention. Therefore,
in some embodiments the culture medium of the invention does not
comprise gastrin. In particular, gastrin is not required for
culturing intestinal stem cells or for obtaining intestinal
(crypt-villus or colon crypt) organoids. However, even where
gastrin is not required, it may still be added to the culture
medium without negative effects.
[0109] In a further embodiment, the culture medium of the invention
is supplemented with nicotinamide. Addition of nicotinamide has
been found to improve culture efficiency and lifespan of human
colon organoids. Nicotinamide may be added to the culture medium to
a final concentration of between 1 and 100 mM, between 5 and 50 mM,
or preferably between 5 and 20 mM. For example, nicotinamide may be
added to the culture medium to a final concentration of
approximately 10 mM.
[0110] In a preferred embodiment of the invention, the culture
medium is supplemented with nicotinamide and gastrin (or a suitable
alternative, such as Leu15-gastrin), wherein nicotinamide and
gastrin are added to the culture medium at any of the
concentrations described above.
[0111] In some embodiments, the culture medium is supplemented with
an activator of the prostaglandin signalling pathway (see FIG. 24,
Antagonism of the prostaglandin D.sub.2 receptors DP.sub.1 and
CRTH2 as an approach to treat allergic diseases. Roy Pettipher,
Trevor T. Hansel & Richard Armer Nature Reviews Drug Discovery
6, 313-325 (April 2007)). For example, the culture medium is
supplemented with any one or more of the compounds selected from
the list comprising: Phospholipids, Arachidonic acid (AA),
prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2
(PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For
example, in some embodiments, the culture medium is supplemented
with PGE2 and/or AA. In some embodiments, PGE2 is added to the
medium to a final concentration of at least 10 nM, for example at
least 20 nM, at least 30 nM, at least 40 nM, at least 45 nM,
between 10 nM and 500 nM, between 10 nM, and 400 nM, between 10 nM
and 300 nM, between 10 nM and 200 nM, between 10 nM and 100 nM,
between 20 nM and 50 nM. In a preferred embodiment, PGE2 is added
to the medium to a final concentration of 50 nM. In some
embodiments, AA is added to the medium to a final concentration of
at least 1 ug/ml, at least 5 ug/ml, at least 8 ug/ml, at least 9
ug/ml, at least 10 ug/ml, for example between 1 ug/ml and 1000
ug/ml, between 1 ug/ml and 500 ug/ml, between 1 ug/ml and 100
ug/ml, between 1 ug/ml and 50 ug/ml, or between 5 ug/ml and 20
ug/ml. In a preferred embodiment, AA is added to the medium to a
final concentration of 10 ug/ml. AA and PGE2 are interchangeable in
the context of the culture media of the invention. Therefore, where
a culture medium described herein is said to include PGE2, it may
alternatively include AA (at an appropriate concentration) instead
of PGE2. Conversely, where a culture medium described herein is
said to include AA, it may alternatively include PGE2 (at an
appropriate concentration) instead of AA. Furthermore, the skilled
person would understand that where PGE2 and/or AA are included in a
culture medium of the invention, the culture medium could instead
comprise any one or more of the compounds selected from the
following list in replacement or in addition to PGE2 and/or AA:
Phospholipids, prostaglandin G2 (PGG2), prostaglandin F2 (PGF2),
prostaglandin H2 (PGH2), and prostaglandin D2 (PGD2).
[0112] In a further embodiment, the culture medium of the invention
is supplemented with RANK ligand (also referred to herein as
RANKL). RANK ligand can be useful for directing differentiation
towards particular cell fates. For example, when RANK ligand is
included in the culture medium for small intestinal cells,
preferably in the medium for differentiating small intestinal
cells, it results in a greater proportion of the cells being
differentiated into M cells. Therefore, in some embodiments, the
invention provides a culture medium comprising RANKL. In
particular, the invention provides a culture medium for culturing,
preferably for differentiating small intestinal cells, wherein the
culture medium comprises RANKL. Any suitable concentration of the
RANKL may be used, for example, between 10 ng/ml and 1000 ng/ml,
between 10 and 500 ng/ml, or between 50 and 100 ng/ml. For example,
RANKL may be added to the culture medium to a final concentration
of approximately 100 ng/ml.
[0113] A culture medium comprising EGF, Noggin and R-spondin is
referred to herein as the "ENR medium". A culture medium comprising
the ENR medium and a Wnt agonist such as Wnt-3a is referred to
herein as the "WENR medium". In a preferred embodiment of the
invention, the culture medium comprises a WENR medium. In a most
preferred embodiment of the invention, the culture medium comprises
a WENR medium supplemented with gastrin and/or nicotinamide (i.e.,
WENRg or WENR+nicotinamide or WENRg+ nicotinamide).
[0114] The pH of the medium may be in the range from about 7.0 to
7.8, in the range from about 7.2 to 7.6, or about 7.4. The pH may
be maintained using a buffer. A suitable buffer can readily be
selected by the skilled person. Buffers that may be used include
carbonate buffers (e.g. NaHCO.sub.3), and phosphates (e.g.
NaH.sub.2PO.sub.4). These buffers are generally used at about 50 to
about 500 mg/l. Other buffers such as
N-[2-hydroxyethyl]-piperazine-N-[2-ethanesul-phonic acid] (HEPES)
and 3-[N-morpholino]-propanesulfonic acid (MOPS) may also be used,
normally at around 1000 to around 10,000 mg/l. A culture medium may
comprise a pH indicator, such as phenol red, to enable the pH
status of the medium to be easily monitored (e.g. at about 5 to
about 50 mg/litre).
[0115] A culture medium for use in the invention may comprise one
or more amino acids. The skilled person understands the appropriate
types and amounts of amino acids for use in stem cell culture
media. Amino acids which may be present include L-alanine,
L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine,
L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine,
L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline,
L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and
combinations thereof. Some culture media will contain all of these
amino acids. Generally, each amino acid when present is present at
about 0.001 to about 1 g/L of medium (usually at about 0.01 to
about 0.15 g/L), except for L-glutamine which is present at about
0.05 to about 1 g/L (usually about 0.1 to about 0.75 g/L). The
amino acids may be of synthetic origin.
[0116] A culture medium for use in the invention may comprise one
or more vitamins. The skilled person understands the appropriate
types and amounts of vitamins for use in stem cell culture media.
Vitamins which may be present include thiamine (vitamin B1),
riboflavin (vitamin B2), niacin (vitamin B3), D-calcium
pantothenate (vitamin B5), pyridoxal/pyridoxamine/pyridoxine
(vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin
B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-alpha
tocopherol (vitamin E), biotin (vitamin H) and menadione (vitamin
K).
[0117] A culture medium for use in the invention may comprise one
or more inorganic salts. The skilled person understands the
appropriate types and amounts of inorganic salts for use in stem
cell culture media. Inorganic salts are typically included in
culture media to aid maintenance of the osmotic balance of the
cells and to help regulate membrane potential. Inorganic salts
which may be present include salts of calcium, copper, iron,
magnesium, potassium, sodium, zinc. The salts are normally used in
the form of chlorides, phosphates, sulphates, nitrates and
bicarbonates.
[0118] Specific salts that may be used include CaCl.sub.2,
CuSO.sub.4-5H.sub.2O, Fe(NO.sub.3)-9H.sub.2O, FeSO.sub.4-7H.sub.2O,
MgCl, MgSO.sub.4, KCl, NaHCO.sub.3, NaCl, Na.sub.2HPO.sub.4,
Na.sub.2HPO.sub.4--H.sub.2O and ZnSO.sub.4-7H.sub.2O.
[0119] The osmolarity of the medium may be in the range from about
200 to about 400 mOsm/kg, in the range from about 290 to about 350
mOsm/kg, or in the range from about 280 to about 310 mOsm/kg. The
osmolarity of the medium may be less than about 300 mOsm/kg (e.g.
about 280 mOsm/kg).
[0120] A culture medium for use in the invention may comprise a
carbon energy source, in the form of one or more sugars. The
skilled person understands the appropriate types and amounts of
sugars to use in stem cell culture media. Sugars which may be
present include glucose, galactose, maltose and fructose. The sugar
is preferably glucose, particularly D-glucose (dextrose). A carbon
energy source will normally be present at between about 1 and about
10 g/L.
[0121] A culture medium of the invention may contain serum. Serum
obtained from any appropriate source may be used, including fetal
bovine serum (FBS), goat serum or human serum. Preferably, human
serum is used. Serum may be used at between about 1% and about 30%
by volume of the medium, according to conventional techniques.
[0122] In other embodiments, a culture medium of the invention may
contain a serum replacement. Various different serum replacement
formulations are commercially available and are known to the
skilled person. Where a serum replacement is used, it may be used
at between about 1% and about 30% by volume of the medium,
according to conventional techniques.
[0123] In other embodiments, a culture medium of the invention may
be serum-free and/or serum replacement-free. A serum-free medium is
one that contains no animal serum of any type. Serum-free media may
be preferred to avoid possible xeno-contamination of the stem
cells. A serum replacement-free medium is one that has not been
supplemented with any commercial serum replacement formulation.
[0124] In a preferred embodiment, the cell culture medium is
supplemented with a purified, natural, semi-synthetic and/or
synthetic growth factor and does not comprise an undefined
component, such as fetal bovine serum or fetal calf serum. For
example, supplements such as B27 (Invitrogen), N-Acetylcysteine
(Sigma) and N2 (Invitrogen) stimulate proliferation of some cells.
In some embodiments, the cell culture medium is supplemented with
one or more of these supplements, for example one, any two or all
three of these supplements.
[0125] In other embodiments, the cell culture medium is
supplemented with Exendin-4. Exendin-4, a 39 amino acid peptide,
activates GLP-1 (glucagon-like peptide-1) receptors to increase
intracellular cAMP in pancreatic acinar cells and has no effect on
VIP (vasoactive intestinal peptide) receptors.
[0126] A culture medium for use in the invention may comprise one
or more trace elements, such as ions of barium, bromium, cobalt,
iodine, manganese, chromium, copper, nickel, selenium, vanadium,
titanium, germanium, molybdenum, silicon, iron, fluorine, silver,
rubidium, tin, zirconium, cadmium, zinc and/or aluminium.
[0127] The medium may comprise a reducing agent, such as
beta-mercaptoethanol at a concentration of about 0.1 mM.
[0128] A culture medium of the invention may comprise one or more
additional agents, such as nutrients or growth factors previously
reported to improve stem cell culture, such as
cholesterol/transferrin/albumin/insulin/progesterone, putrescine,
selenite/other factors.
[0129] A culture medium of the invention may be diffused into an
extracellular matrix (ECM). In a preferred method of the invention,
isolated tissue fragments or isolated epithelial stem cells are
attached to an ECM. ECM is composed of a variety of
polysaccharides, water, elastin, and glycoproteins, wherein the
glycoproteins comprise collagen, entactin (nidogen), fibronectin,
and laminin. ECM is secreted by connective tissue cells. Different
types of ECM are known, comprising different compositions including
different types of glycoproteins and/or different combination of
glycoproteins. Said ECM can be provided by culturing ECM-producing
cells, such as for example fibroblast cells, in a receptacle, prior
to the removal of these cells and the addition of isolated tissue
fragments or isolated epithelial stem cells. Examples of
extracellular matrix-producing cells are chondrocytes, producing
mainly collagen and proteoglycans, fibroblast cells, producing
mainly type IV collagen, laminin, interstitial procollagens, and
fibronectin, and colonic myofibroblasts producing mainly collagens
(type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic
acid, fibronectin, and tenascin-C. Alternatively, said ECM is
commercially provided. Examples of commercially available
extracellular matrices are extracellular matrix proteins
(Invitrogen) and basement membrane preparations from
Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g. Matrigel.TM.
(BD Biosciences)). A synthetic extracellular matrix material, such
as ProNectin (Sigma Z378666) may be used. Mixtures of extracellular
matrix materials may be used, if desired. The use of an ECM for
culturing stem cells enhanced long-term survival of the stem cells
and the continued presence of undifferentiated stem cells. In the
absence of an ECM, stem cell cultures could not be cultured for
longer periods and no continued presence of undifferentiated stem
cells was observed. In addition, the presence of an ECM allowed
culturing of three-dimensional tissue organoids, which could not be
cultured in the absence of an ECM. The extracellular matrix
material will normally be a drop on the bottom of the dish in which
cells are suspended. Typically, when the matrix solidifies at
37.degree. C., the medium is added and diffuses into the ECM. The
cells in the medium stick to the ECM by interaction with its
surface structure, for example interaction with integrins. A
fibronectin solution of about 1 mg/ml (stock solution) used at
approximately 1 .mu.g/cm.sup.2 may be used to coat a cell culture
vessel, or between about 1 .mu.g/cm.sup.2 to about 250
.mu.g/cm.sup.2, or at about 1 .mu.g/cm.sup.2 to about 150
.mu.g/cm.sup.2. In some embodiments, a cell culture vessel is
coated with fibronectin at between 8 .mu.g/cm.sup.2 and 125
.mu.g/cm.sup.2.
[0130] An example of an ECM for use in a method of the invention
comprises at least one glycoprotein, such as laminin.
[0131] A preferred ECM for use in a method of the invention
comprises at least two distinct glycoproteins, such as two
different types of collagen or a collagen and laminin. The ECM can
be a synthetic hydrogel extracellular matrix or a naturally
occurring ECM. A further preferred ECM is provided by Matrigel.TM.
(BD Biosciences), which comprises laminin, entactin, and collagen
IV. In some embodiments the extracellular matrix is a
laminin-containing extracellular matrix such as Matrigel.TM. (BD
Biosciences).
[0132] In some embodiments, the single stem cell, population of
cells, or tissue fragment is embedded in matrigel, which is
optionally growth factor reduced and/or phenol red-free.
[0133] In some embodiments, the culture medium is placed on top of
the ECM. The culture medium can then be removed and replenished as
and when required. In some embodiments, the culture medium is
replenished every 1, 2, 3, 4, 5, 6 or 7 days. If components are
"added" or "removed" from the media, then this can in some
embodiments mean that the media itself is removed from the ECM and
then a new media containing the "added" component or with the
"removed" component excluded is placed on the ECM.
[0134] In some embodiments the culture medium of the invention is
in contact with an extracellular matrix or a 3D matrix that mimics
the extracellular matrix by its interaction with the cellular
membrane proteins, such as integrins.
[0135] In some embodiments, the basal culture medium comprises or
consists of Advanced DMEM/F12 supplemented with
penicillin/streptomycin, 10 mM HEPES, Glutamax, 1.times.N2,
1.times.B27 (all from Invitrogen) and 1 mM N-acetylcysteine
(Sigma)).
Examples of Culture Media of the Invention
[0136] In one embodiment, the cell culture medium comprises a
TGF-beta inhibitor that binds to and reduces the activity of ALK5
and a p38 inhibitor that binds to and reduces the activity of p38.
For example, in one embodiment the cell culture media comprises
A83-01 and/or SB202190, preferably A83-01+SB202190. The use of
A83-01+SB202190 together in a culture medium of the invention has
surprisingly be found to synergistically increase the number of
passages of human colon organoids. In one embodiment, the cell
culture media comprises WENR+A83-01+SB202190. In one embodiment,
the cell culture media comprise WENR+A83-01+SB202190+ nicotinamide.
In one embodiment, the cell culture media comprises WENRg+
nicotinamide+A83-01+SB202190 (where "g" is gastrin). In one
embodiment, the cell culture medium comprises
WENR+A83-01+Nicotinamide+FGF10. In one embodiment, the cell culture
medium comprises WENRg+A83-01+Nicotinamide+FGF10. In one
embodiment, the cell culture medium comprises
WENRg+A83-01+Nicotinamide+FGF10+SB202190. In one embodiment, the
cell culture media is used for obtaining colon organoids. A colon
organoid obtainable by culturing epithelial cells using a cell
culture media as described in this embodiment is also provided.
[0137] For example, in one embodiment the cell culture media
comprises WENRg+A83-01+FGF10, wherein the Wnt agonist is R-Spondin
but no other Wnt agonist is present and no nicotinamide is present.
For example, in some embodiments, the cell culture media comprises
EGF (e.g. 50 ng/ml), R-Spondin (e.g. 10% or 1 ug/ml), Noggin (e.g.
100 ng/ml), FGF10 (e.g. 100 ng/ml), A8301 (e.g. 500 nM) and Gastrin
(e.g. 10 uM) and optionally SB202190. These components may be added
to a basal medium, such as DMEM/F12 media. In some embodiments, the
basal medium is further supplemented with any one or more (for
example, 1, 2, 3, 4 or 5) or all, of the components selected from
the list comprising: P/S, Glutamax, 10 nmM Hepes, B27, N2 and
N-Acetylcysteine. The use of such a cell culture media has been
found to be useful for obtaining pancreatic organoids. A pancreatic
organoid obtained by culturing epithelial cells using a cell
culture media as described in this embodiment is also provided. In
some embodiments, gastrin or nicotinamide or gastrin and
nicotinamide are excluded from the culture medium.
Tissue-Specific Culture Media of the Invention
[0138] Particularly preferred culture media are described in the
Examples herein. The culture medium of the invention can be adapted
for use with different tissues, for example as described below.
Intestinal Culture Media
[0139] In some embodiments, the culture medium for small intestinal
crypts, such as murine small intestinal crypts, comprises or
consists of a basal medium, for example as described above,
additionally comprising: EGF, such as murine EGF; a BMP inhibitor,
such as murine Noggin; and Rspondin, such as human Rspondin-1 or 4.
In some embodiments, this culture medium further comprises a
TGF-beta inhibitor (such as A83-01) and/or a p38 inhibitor (such as
SB202190). In some embodiments, the culture medium for colonic
crypts, such as murine colonic crypts, comprises or consists of a
basal medium, for example as described above, additionally
comprising: a Wnt agonist, such as recombinant human Wnt-3A or
Wnt-3A conditioned medium; EGF, such as murine EGF; a BMP
inhibitor, such as murine Noggin; and Rspondin, such as human
Rspondin-1 or 4. In some embodiments, this culture medium further
comprises a TGF-beta inhibitor (such as A83-01) and/or a p38
inhibitor (such as SB202190).
[0140] In some embodiments, the culture medium for human intestinal
stem cells, human small intestinal crypts or human colonic crypts
(also known as the HISC culture medium), comprises or consists of a
basal medium, for example as described above, additionally
comprising: a Wnt agonist, such as recombinant human Wnt-3A or
Wnt-3A conditioned medium; EGF; a BMP inhibitor, such as Noggin;
Rspondin, such as human Rspondin-1; a TGF-beta inhibitor, such as
A83-01; a p38 inhibitor, such as SB202190; gastrin; and
nicotinamide. In some embodiments, the p38 inhibitor and/or gastrin
can be excluded from the HISC culture medium.
[0141] In some embodiments the invention provides a culture medium
for culturing intestinal cells, comprising or consisting of a basal
medium, Wnt-3a, EGF, Noggin, any one of Rspondin 1-4, a TGF-beta
inhibitor, nicotinamide, and preferably a p38 inhibitor.
[0142] In some embodiments, the culture medium for expanding small
intestine or colon stem cells, for example human small intestine or
colon cells, comprises or consists of a basal medium (for example
comprising Advanced DMEM/F12, B27 (50.times.), n-Acetylcysteine (1
mM) and glutamin/glutamax), Wnt3A (optionally conditioned medium),
any one of Rspondin 1-4 (preferably 1 ug/ml), Noggin (preferably
50-100 ng/ml), nicotinamide (preferably 10 mM), EGF (preferably
10-50 ng/ml), gastrin (preferably 10 nM), a TGF-beta inhibitor, for
example A83-01 (preferably 500 nM). In a further embodiment, this
culture medium additionally comprises a p38 inhibitor, for example
SB202190 (preferably 100 nM). In a further embodiment, this culture
medium additionally comprises a Rock inhibitor, for example
LY2157299.
[0143] In some embodiments, the invention provides a culture medium
for differentiating intestinal cells, comprising or consisting of a
basal medium, EGF, Noggin, a TGF-beta inhibitor and a p38
inhibitor.
[0144] In some embodiments, the culture medium for differentiating
small intestine or colon stem cells, for example human small
intestine or colon cells, comprises or consists of a basal medium
(for example comprising Advanced DMEM/F12, B27 (50.times.),
n-Acetylcysteine (1 mM) and glutamin/glutamax), Noggin (preferably
50-100 ng/ml), EGF (preferably 10-50 ng/ml), gastrin (preferably 10
nM), a TGF-beta inhibitor, for example A83-01 (preferably 500 nM)
and a p38 inhibitor, for example SB202190 (preferably 100 nM). In
some embodiments, gastrin can be excluded from this differentiation
medium. In some embodiments, a gamma-secretase inhibitor may be
added to the differentiation medium (preferably at a concentration
of 1 uM). Gamma-secretase inhibitors can influence cell fate
decisions during differentiation e.g. towards secretory cells, such
as goblet cells. In some embodiments, a RANKL may be added to the
differentiation medium (for example at a concentration of 100
ng/ml). RANKL can influence cell fate decisions during
differentiation e.g. towards M-cells.
Cancer Culture Media
[0145] In some embodiments, the culture medium for colon cancer
cells, comprises or consists of a basal medium, for example as
described above, additionally comprising: a Wnt agonist, such as
recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP
inhibitor, such as Noggin; Rspondin, such as human Rspondin-1; a
TGF-beta inhibitor, such as A83-01; a p38 inhibitor, such as
SB202190; gastrin; and nicotinamide.
[0146] In one embodiment, the culture medium for colon carcinoma,
for example human colon carcinoma, comprises a basal medium (for
example comprising Advanced DMEM/F12, B27 (50.times.),
n-Acetylcysteine (1 mM), primocin and/or P/S (antibiotics)
(500.times.) and hepes), Rspondin (optionally conditioned medium)
(preferably 1 ug/ml), Noggin (preferably 100 ng/ml), Nicotinamide
(preferably 10 mM), EGF (preferably 50 ng/ml), gastrin (preferably
50 nM), a TGF-beta inhibitor, for example A83-01 (preferably 500
nM), a p38 inhibitor, such as SB202190 (preferably 10 uM),
optionally PGE2 (preferably 10 nM) and/or a Rock inhibitor
(preferably 10 uM).
[0147] In some embodiments, colon cancer cells can also be grown in
the HISC culture medium. In some embodiments, colon cancer cells
can be cultured in the HISC culture medium, wherein one or more or
all of the following are excluded from the medium: EGF, Noggin,
Rspondin, TGF-beta inhibitor and p38 inhibitor. Cancer cells may
have mutations that consitutively activate or deactivate certain
growth pathways. For example, many colon cancers result in
constitutive activation of the Wnt pathway. In such cases, a
culture medium would not require a Wnt agonist. Other mutations
would allow other factors to be left out of the medium as described
above. Other epithelial cancers (carcinomas) can also be grown in
culture media of the invention. In a preferred embodiment, a cancer
organoid obtained from cancer stem cells is grown in a culture
medium that is suitable for growth of the corresponding normal
tissue organoid obtained from normal stem cells, optionally with
certain factors excluded from the medium. For example, a stomach
cancer organoid obtained by culturing stomach cancer stem cells may
be grown in the same culture conditions as a normal gastric
organoid obtained by culturing gastric stem cells, optionally with
certain factors excluded from the medium. In another example, a
pancreatic cancer organoid obtained by culturing pancreatic cancer
stem cells may be grown in the same culture conditions as a normal
pancreatic organoid obtained by culturing pancreatic stem cells,
optionally with certain factors excluded from the medium. In
another example, a prostate cancer organoid obtained by culturing
prostatic cancer stem cells may be grown in the same culture
conditions as a normal prostate organoid obtained by culturing
prostatic stem cells, optionally with certain factors excluded from
the medium. In another example, a liver cancer organoid obtained by
culturing liver cancer stem cells may be grown in the same culture
conditions as a normal liver organoid obtained by culturing liver
stem cells, optionally with certain factors excluded from the
medium. In many situations it may be preferable (or at least more
convenient) to grow cancer organoids in the normal tissue medium
(without any factors excluded). The normal tissue medium should
allow cancers with all genetic backgrounds to grow, without
excluding any particular cancer mutations.
[0148] Therefore, in some embodiments, the invention provides a
culture medium for culturing cancer cells, for example cancer stem
cells, such as adenocarcinoma or carcinoma cells from a tissue type
of interest, wherein the culture medium comprises or consists of
the components of the culture medium used for culturing the cells
from the corresponding non-cancerous tissue type of interest,
optionally wherein one or more of the following are excluded from
the medium that is used to culture the non-cancerous cells of the
tissue type of interest: Wnt-3a, EGF, Noggin, Rspondin, TGF-beta
inhibitor, p38 inhibitor, nicotinamide, gastrin, FGF10 and HGF.
Adenoma Culture Medium
[0149] In some embodiments, the culture medium for intestinal
adenomas, such as murine intestinal adenomas comprises a basal
medium, for example as described above, additionally comprising
EGF, such as murine EGF.
Gastric Culture Media
[0150] In some embodiments, the invention provides a culture medium
for culturing gastric cells, comprising or consisting of a basal
medium, Wnt-3 a, EGF, Noggin, any one of Rspondin 1-4, a TGF-beta
inhibitor, gastrin, nicotinamide, FGF-10, and preferably a p38
inhibitor.
[0151] In some embodiments, the culture medium for gastric stem
cells, for example human gastric stem cells comprises or consists
of a basal medium (for example comprising Advanced DMEM/F12, B27
(50.times.), n-Acetylcysteine (1 mM), primocin and/or P/S
(antibiotics) (500.times.) and glutamin/glutamax), any one of
Rspondin 1-4 (optionally conditioned medium) (preferably 1 ug/ml),
Noggin (optionally conditioned medium) (preferably 100 ng/ml),
Wnt3A (optionally conditioned medium), nicotinamide (preferably 5
mM), EGF (preferably 50 ng/ml), FGF10 (preferably 200 ng/ml),
gastrin (preferably 1 nM), a TGF-beta inhibitor, for example A83-01
(preferably 2 uM). The culture medium for gastric stem cells
optionally further comprises a p38 inhibitor, for example SB202190
(preferably 10 nM). The culture medium for gastric stem cells
optionally further comprises PGE2 (preferably 500 nM). The culture
medium for gastric stem cells optionally further comprises a Rock
inhibitor (preferably 10 uM).
[0152] In some embodiments, the culture medium for gastric stem
cells, for example murine gastric cells comprises or consists of a
basal medium (for example comprising Advanced DMEM/F12, B27
(50.times.), n-Acetylcysteine (1 mM), primocin and/or P/S
(antibiotics) (500.times.) and glutamin/glutamax), any one of
Rspondin 1-4 (optionally conditioned medium) (preferably 1 ug/ml),
Noggin (optionally conditioned medium) (preferably 100 ng/ml),
Wnt3A (optionally conditioned medium), EGF (preferably 50 ng/ml),
FGF10 (preferably 200 ng/ml), gastrin (preferably 1 nM) and a Rock
inhibitor (preferably 10 uM). In some embodiments, this culture
medium further comprises a TGF-beta inhibitor (such as A83-01)
and/or a p38 inhibitor (such as SB202190).
Prostate Culture Media
[0153] In some embodiments, the culture medium for expanding
prostate stem cells comprises testosterone, optionally
dihydrotestosterone (also referred to herein as DHT). Testosterone
is a steroid hormone from the androgen group. In humans, a large
percentage of testosterone undergoes 5.alpha.-reduction to form the
more potent androgen, dihydrotestosterone. Testosterone,
dihydrotestosterone or a testosterone mimic (for example, a
molecule that mimics the activity of testosterone binding to an
androgen receptor) can be added to a culture medium of the
invention. Therefore, where the term testosterone is used, it can
always be replaced by dihydrotestosterone or a testosterone mimic.
The inventors have shown that addition of testosterone to a culture
medium for prostate stem cells, results in increased
differentiation but also in continued expansion of the stem cell
population (for example, see FIGS. 41-45). This is highly
surprising because the literature teaches that testosterone plays
an important role in the differentiation of cells by acting to
suppress proliferation and maintain terminal differentiation
(Mirochnik et al. PLoS One, 7(3), e31052, 2012; Niu et al. Oncogene
29, 3593-3604, 2010). The skilled person would have expected that
addition of testosterone to a culture medium for prostate would
result in completely differentiated organoids with no further
expansion potential. This would be similar to what is observed when
the colon, pancreas and liver organoids are differentiated in a
differentiation medium. However, by contrast, the present inventors
have found that although testosterone increases differentiation, it
also allows stem cell expansion to continue. Therefore, organoids
grown in a culture medium comprising testosterone surprisingly
comprise stem cells and differentiated cells i.e. luminal cells and
basal cells.
[0154] In some embodiments, the culture medium for obtaining a
prostate organoid comprises a basal medium and testosterone,
optionally dihydrotestosterone and anyone of Rspondin 1-4 or an
Rspondin mimic. In some embodiments, the culture medium further
comprises a BMP inhibitor, for example Noggin. In some embodiments,
the culture medium further comprises a tyrosine receptor kinase
ligand, optionally wherein the tyrosine receptor kinase ligand is a
mitogenic growth factor, such as EGF, FGF, KGF or HGF. In some
embodiments, the culture medium for obtaining a prostate organoid
comprises EGF, Noggin, any one or Rspondin 1-4 and
testosterone.
[0155] In a preferred embodiment, the culture medium for prostate
cells comprises a TGF-beta inhibitor. In some embodiments, the
culture medium for prostate cells comprises EGF, Noggin, any one of
Rspondin 1-4, a TGF-beta inhibitor and testosterone. In some
embodiments, the culture medium for prostate cells further
comprises a p38 inhibitor. In some embodiments, a culture medium
for obtaining a prostate organoid does not comprise an inhibitor of
the invention, for example a TGF-beta inhibitor and/or a p38
inhibitor. In some embodiments the culture medium for prostate stem
cells does not comprise testosterone. In some embodiments, the
culture medium comprises a basal medium, EGF, Noggin and any one of
Rspondin 1-4 and optionally a TGF-beta inhibitor, and does not
comprise testosterone.
[0156] In some embodiments, the invention provides a culture medium
for culturing prostate cells, comprising or consisting of a basal
medium, EGF, any one of Rspondin 1-4, Noggin, nicotinamide a
TGF-beta inhibitor, and preferably Wnt-3a and FGF-10. In some
embodiments, the culture medium for culturing prostate cells
further comprises testosterone, for example (dihydro)testosterone.
In some embodiments the culture medium further comprises a p38
inhibitor. In some embodiments, the culture medium for prostate
cells, for example mouse, human, normal or carcinoma, comprises a
basal medium (for example comprising Advanced DMEM/F12, B27
(50.times.), n-Acetylcysteine (1 mM) and glutamin/glutamax), any
one of Rspondin 1-4 (optionally conditioned medium) (preferably 1
ug/ml), Noggin (optionally conditioned medium) (preferably 100
ng/ml), nicotinamide (preferably 10 mM), EGF (preferably 50 ng/ml),
FGF10 (preferably 100 ng/ml), a TGF-beta inhibitor, for example
A83-01 (preferably 500 nM), (Dihydro)testosterone (preferably 1 nM)
10 nM and optionally Wnt-3a. In some embodiments, this culture
medium further comprises a Rock inhibitor (preferably 10 uM). In
some embodiments, the culture medium for prostate cells further
comprises a p38 inhibitor, for example SB202190. In some
embodiments, wherein mouse prostate cells are cultured, the
TGF-beta inhibitor can be excluded from the culture medium. In
other embodiments, nicotinamide, FGF 10 and/or the Rock inhibitor
can be excluded from the culture medium.
Pancreatic Culture Media
[0157] In some embodiments, the invention provides a culture medium
for expanding pancreas cells, comprising or consisting of a basal
medium, any one of Rspondin 1-4, Noggin, EGF, FGF10, gastrin, a
TGF-beta inhibitor, and preferably exendin 4 and Wnt-3a.
[0158] In some embodiments, the culture medium for expanding
pancreatic stem cells, for example human pancreatic stem cells
comprises or consists of a basal medium (for example comprising
Advanced DMEM/F12, B27 (50.times.), n-Acetylcysteine (1 mM) and
glutamin/glutamax), any one of Rspondin 1-4 (optionally conditioned
medium) (preferably 1 ug/ml), Noggin (optionally conditioned
medium) (preferably 100 ng/ml), nicotinamide (preferably 10 mM),
EGF (preferably 50 ng/ml), FGF10 (preferably 100 ng/ml), gastrin
(preferably 100 nM), and a TGF-beta inhibitor, for example A83-01
(preferably 2 uM). In a further embodiment, this culture medium
additionally comprises Wnt-3a. In a further embodiment, this
culture medium additionally comprises a p38 inhibitor, for example
SB202190 (preferably 100 nM). In a further embodiment, this culture
medium additionally comprises a Rock inhibitor, for example
LY2157299 (preferably 10 uM). In a further embodiment, this culture
medium additionally comprises Exendin 4 (preferably 50 ng/ml).
[0159] In some embodiments, the culture medium for expanding
pancreatic stem cells, for example mouse pancreatic stem cells
comprises or consists of a basal medium (for example comprising
Advanced DMEM/F12, B27 (50.times.), n-Acetylcysteine (1 mM),
primocin and/or P/S (antibiotics), Hepes and glutamin/glutamax),
any one of Rspondin 1-4 (optionally conditioned medium) (preferably
1 ug/ml), Noggin (optionally conditioned medium) (preferably 100
ng/ml), nicotinamide (preferably 10 mM), EGF (preferably 50 ng/ml),
FGF10 (preferably 100 ng/ml), gastrin (preferably 100 nM), and a
TGF-beta inhibitor, for example A83-01 (preferably 2 uM). In a
further embodiment, this culture medium additionally comprises a
Rock inhibitor, for example LY2157299 (preferably 10 uM). In some
embodiments, the culture medium for pancreatic cells further
comprises a p38 inhibitor, for example SB202190.
[0160] In some embodiments, the invention provides a culture medium
for differentiating pancreas cells comprising or consisting of a
basal medium, Noggin, EGF, FGF10, gastrin, a TGF-beta inhibitor,
gamma-secretase inhibitor and preferably exendin 4.
[0161] In some embodiments, the culture medium for differentiating
pancreatic stem cells, for example human pancreatic stem cells
comprises or consists of a basal medium (for example comprising
Advanced DMEM/F12, B27 (50.times.), n-Acetylcysteine (1 mM) and
glutamin/glutamax), Noggin (preferably 100 ng/ml), EGF (preferably
50 ng/ml), FGF10 (preferably 10 nM), gastrin (preferably 100 nM), a
TGF-beta inhibitor, for example A83-01 (preferably 50 nM) and
gamma-secretase inhibitor (DAPT/DBZ) (preferably 10 uM). In a
further embodiment, this culture medium additionally comprises
Exendin 4 (preferably 50 ng/ml). In some embodiments, the culture
medium for pancreatic cells further comprises a p38 inhibitor, for
example SB202190.
[0162] In some embodiments, the culture medium for differentiating
pancreatic stem cells, for example mouse pancreatic stem cells
comprises or consists of a basal medium (for example comprising
Advanced DMEM/F12, B27 (50.times.), n-Acetylcysteine (1 mM) and
glutamin/glutamax), EGF (preferably 50 ng/ml) and gamma-secretase
inhibitor (for example DAPT/DBZ) (preferably 10 uM).
Barrett's Esophagus Culture Medium
[0163] In some embodiments, the culture medium for Barrett's
Esophagus, comprises or consists of a basal medium, for example as
described above, additionally comprising: a Wnt agonist, such as
recombinant human Wnt-3A or Wnt-3A conditioned medium; EGF; a BMP
inhibitor, such as Noggin; Rspondin, such as human Rspondin-1; a
TGF-beta inhibitor, such as A83-01; a p38 inhibitor, such as
SB202190; gastrin; nicotinamide; and an FGF, such as human FGF10
(i.e. HISC+FGF). In some embodiments, gastrin is excluded from this
culture medium.
[0164] In some embodiments, the invention provides a method for
obtaining a Barrett's Esophagus organoid, wherein the method
comprises culturing isolated epithelium from Barrett's Esophagus
for 1, 2, 3, 4, 5, 6, 7 or more days in HISC culture medium,
optionally additionally comprising FGF10; and withdrawing
nicotinamide and SB202190 after the first 1, 2, 3, 4 or more days.
In some embodiments the culture medium additionally comprises a
Notich inhibitor, such as DBZ. In some embodiments, a Barrett's
Esophagus organoid cultured in the presence of a Notch inhibitor
comprises almost no or no proliferating cells, and comprises more
goblet cells relative to an organoid cultured in the absence of a
Notch inhibitor (see FIG. 5).
Liver Culture Media
[0165] In some embodiments, liver cells can be grown in a first
"expansion" culture medium (also referred to herein as EM),
preferably followed by culturing the cells in a second
"differentiation" culture medium (also referred to herein as DM).
However, in some embodiments, the step of differentiating in DM
media is not carried out, for example in some methods, cells are
transplanted and allowed to differentiate in vivo. Similarly, there
are expansion culture media and differentiation culture media for
other tissues, such as the pancreas, small intestine and colon (see
above).
[0166] In one embodiment, the liver expansion medium comprises EGF,
a Wnt agonist, FGF, and Nicotinamide. Preferably, the Wnt agonist
is R-spondin 1-4 (for example any one or more of Rspondin 1, 2, 3,
and 4) and so the expansion medium is referred to as "ERFNic". A
particularly preferred expansion medium additionally comprises HGF
and is referred to as "ERFHNic".
[0167] In some embodiments, the liver expansion medium is
supplemented with a TGF beta inhibitor. In some embodiments, TGF
beta is present at at least 5 nM, for example, at least 50 nM, at
least 100 nM, at least 300 nM, at least 450 nM, at least 475 nM,
for example 5 nM-500 mM, 10 nM-100 mM, 50 nM-700 uM, 50 nM-10 uM,
100 nM-1000 nM, 350-650 nM or more preferably 500 nM. The presence
of a TGF beta inhibitor in the expansion media is particularly
preferred for human cell embodiments.
[0168] In some embodiments, the invention provides a culture medium
for expanding liver cells, comprising or consisting of a basal
medium, any one of Rspondin 1-4, Noggin, nicotinamide, EGF, FGF10,
HGF, gastrin, a TGF-beta inhibitor and PGE2, and preferably
Wnt-3a.
[0169] In some embodiments, the liver expansion medium further
comprises a p38 inhibitor.
[0170] In some embodiments, the liver expansion medium is
supplemented with an activator of the prostaglandin signalling
pathway (also called a prostaglandin pathway activator) (see FIG.
24). For example, the liver expansion medium may be supplemented
with any one or more of the compounds selected from the list
comprising: Phospholipids, Arachidonic acid (AA), prostaglandin E2
(PGE2), prostaglandin G2 (PGG2), prostaglandin F2 (PGF2),
prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For example, in
some embodiments, the liver expansion medium is supplemented with
PGE2 and/or AA. In some embodiments, PGE2 is added to the liver
expansion medium to a final concentration of at least 10 nM, at
least 30 nM, at least 40 nM, at least 45 nM, at least 50 nM, for
example between 10 nM and 500 nM, between 10 nM and 400 nM, between
10 nM and 300 nM, between 10 nM and 200 nM, between 10 nM and 100
nM, between 20 nM and 50 nM. In a preferred embodiment, PGE2 is
added to the liver expansion medium to a final concentration of 50
nM. In some embodiments, AA is added to the liver expansion medium
to a final concentration of at least 1 ug/ml, for example at least
3 ug/ml, at least 5 ug/ml, at least 8 ug/ml, at least 9 ug/ml, at
least 10 ug/ml, between 1 ug/ml and 1000 ug/ml, between 1 ug/ml and
500 ug/ml, between 1 ug/ml and 100 ug/ml, between 1 ug/ml and 50
ug/ml, or between 5 ug/ml and 10 ug/ml. In a preferred embodiment,
AA is added to the medium to a final concentration of 10 ug/ml.
[0171] In a preferred embodiment, the liver expansion medium is
supplemented with both a TGF-beta inhibitor and an activator of the
prostaglandin signalling pathway (for example, PGE2 and/or AA) and
optionally a p38 inhibitor.
[0172] In preferred embodiments, the liver expansion medium
additionally comprises gastrin.
[0173] In one embodiment, the liver differentiation medium
comprises EGF, a TGF-beta inhibitor, FGF (for example, FGF10, FGF2
or any other suitable FGF family member) and a Notch inhibitor. In
one embodiment, the TGF-beta inhibitor is A83-01 and/or the Notch
inhibitor is DAPT. This differentiation medium is referred to
herein as "EAFD" and is a preferred differentiation medium of the
invention. FGF may optionally be replaced by HGF or alternatively
both FGF and HGF may be present or absent in the differentiation
medium. In some embodiments, EGF might be replaced by HGF or
another receptor tyrosine kinase ligand. Dexamethasone may also be
added, for example at a concentration of between 10 nM to 10 uM.
The liver differentiation medium may optionally include a
prostaglandin pathway activator, such as PGE2 or AA. However, this
component may also be excluded from the differentiation medium. In
some embodiments, oncostatin M may also be added, for example at a
concentration range between 1 ng/ml to 1 mg/ml, to help
differentiation to hepatocyte fate.
[0174] In some embodiments, the invention provides a culture medium
for differentiating liver cells comprising or consisting of a basal
medium, Noggin, EGF, gastrin, a TGF-beta inhibitor, a
gamma-secretase inhibitor such as DAPT or DBZ, and preferably
Wnt-3a.
[0175] In some embodiments, the liver cells may initially be
cultured in an expansion medium that additionally contains Wnt and
Noggin, for example an "ENRW" medium containing EGF, Noggin,
R-spondin and Wnt (for example, Wnt-3A), optionally a prostaglandin
pathway activator, such as PGE2 or AA, optionally a TGF beta
inhibitor and optionally FGF, HGF, Nicotinamide. In a preferred
embodiment, the liver expansion medium is supplemented with one of
or more preferably both of a TGF-beta inhibitor and an activator of
the prostaglandin signalling pathway (for example, PGE2 and/or
AA).
[0176] In some embodiments, the expansion media for liver comprises
EGF, Noggin, Gastrin, FGF, Nicotinamide, a TGF-beta inhibitor such
as A83-01, HGF, RSpondin 1-4 (e.g. any one or more of Rspondin 1,
2, 3 and 4) and PGE2.
[0177] In a preferred embodiment, the liver cells may initially be
cultured in an expansion media that contains EGF, noggin, gastrin,
FGF10, nicotinamide, A8301, HGF and any one of Rspondin 1-4
supplemented with PGE2 and/or AA. Rspondin 1-4 may be provided in
the form of Rspo conditioned media. For example, the expansion
media may contain EGF (100 ng/ml, Invitrogen); noggin (25 ng/ml,
peprotech); gastrin (10 nM, sigma); FGF10 (e.g. 100 ng/ml,
peprotech); nicotinamide (10 mM, sigma); A8301 (500 nM, Tocris);
HGF (50 ng/ml, peprotech); Rspo conditioned media (10%, e.g. 1
ug/ml) supplemented with PGE2 (50 nM) and/or AA (10 ug/ml). The
expansion medium may also contain a Rock inhibitor.
[0178] When expanding mouse liver cells, one or more of the
following components may be excluded from the culture medium
described above: TGF-beta inhibitor (e.g. A83-01) and PGE2.
[0179] The inventors have found that this medium is optimal for
stimulating initial expansion of cells for the first few days.
Therefore, this first expansion medium is sometimes referred to
herein as EM1. In some embodiments, the Wnt and Noggin are removed
after approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days,
7 days or more, for example 2 weeks, 1 month, 5 weeks, 8 weeks, 2
months 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, 14 months or
more. In some embodiments, the cells may then be expanded in an
expansion medium of the invention, as described above that does not
contain Wnt or Noggin. This second expansion medium is sometimes
referred to herein as EM2. In some embodiments, the cells are
cultured in EM2 for approximately 1 day, 2 days, 3 days, 4 days, 5
days, 6 days, 7 days or a longer time period, such as 3, 4, 5, 10,
20 or more weeks. The culture medium may then be changed to an
optimised differentiation medium, as described above, that contains
a TGF-beta inhibitor and a Notch inhibitor. Typically, the
differentiation medium does not contain a Wnt agonist, R-spondin or
Nicotinamide. In some embodiments, the differentiation medium does
not contain a prostaglandin pathway activator, such as PGE2 or AA.
This encourages the differentiation of the cells towards mature
hepatocytes and cholangyocytes. These cells are suitable for
transplantation into humans or animals.
Expansion Medium (EM2) for Liver:
[0180] In one aspect of the present invention there is provided a
cell culture medium which comprises or consists of a basal medium
for animal or human cells to which is added: Epidermal Growth
Factor, an FGF able to bind to FGFR2 or FGFR4, preferably FGF10 as
a mitogenic growth factor, Nicotinamide, and preferably, a Wnt
agonist, preferably R-spondin 1-4. This medium is referred to as
EM2. This "EM2" medium is preferred for expanding liver cells.
[0181] In some embodiments, EM2 comprises a prostaglandin pathway
activator such as PGE2 and/or AA.
[0182] In some embodiments, EM2 comprises a TGF-beta inhibitor such
as A83-01.
[0183] Preferably, the Wnt agonist is R-spondin 1-4. A medium
comprising EGF, R-spondin 1-4, FGF and Nicotinamide is referred to
herein as ERFNic.
[0184] In some embodiments, the EM2 medium does not comprise
noggin, and more preferably does not comprise a BMP-inhibitor. In
some embodiments, the EM2 medium does not comprise Wnt, for example
Wnt-3a.
[0185] In some embodiments, HGF is present in addition to FGF. A
preferred medium comprising HGF in addition to FGF is ERFHNic
(EGF+R-spondin (preferably R-spondin1-4)+FGF (preferably
FGF10)+HGF+Nicotinamide+a prostaglandin pathway inhibitor such as
PGE2 and/or AA and a TGF-beta inhibitor. The inventors have found
that the ERFHNic medium containing the TGF-beta inhibitor and the
prostaglandin pathway activator is the optimal medium for long-term
expansion of cells. In the absence of HGF, cells did not remain
viable in culture for longer than three months. Further, in the
absence of HGF, after 10 passages, cells showed a growth
disadvantage compared to cells cultured in the presence of HGF as
evidenced by a lower proliferation ratio. In particular, after 15
passages, the cells were not growing organoids at the same speed
ratio in the absence of HGF as in the presence of HGF. Thus, HGF
was found to be essential for maintaining a good proliferation rate
during long-term culture. Thus the invention provides the use of an
ERFHNic medium of the invention for culturing cells for at least 2
weeks, at least 1 month, at least 2 months, more preferably at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, at least 10, at least 15, at least 20, at least 24,
at least 25, at least 30 or more months, for example 3 or more
years. In practice, some embodiments of the invention comprise the
use of EM2 for around 20-50 passages of the cells. For example, the
cells may be split 4-8 times once a week for 7-10 weeks in a row.
Preferably the cells will expand at a rate of about 4-5 fold per
week or more than two population doublings a week. The invention
further provides the use of an ERFHNic medium of the invention for
culturing cells for at least 8 passages, for example, at least 9,
at least 10, at least 11, at least 12, at least 15, at least 20, at
least 25, at least 30, at least 40, at least 50, at least 60
passages or for between 15-35 passages, for example approximately
20-30 passages. In some embodiments, a TGF-beta inhibitor such as
A83-01 is additionally present in the EM2 medium. This is
particularly useful when human cells or organoids are being
cultured. In some embodiments, the A83-01 is present at a
concentration of between 400-600 nM, for example 450-550 nM,
470-530 nM or approximately 500 nM. In embodiments in which a
TGF-beta inhibitor is present in EM2, a Notch inhibitor is
preferably not present. In some embodiments, EM2 additionally
comprises a p38 inhibitor.
Expansion Medium (EMI) for Liver:
[0186] In one aspect, the invention provides a cell culture medium
comprising or consisting of a basal medium for animal or human
cells to which is added EGF, a BMP inhibitor, R-spondin Wnt.
Preferably, the BMP inhibitor is Noggin and the EM1 medium is
termed "ENRW" (EGF, Noggin, R-spondin and Wnt (e.g. Wnt3A)). This
medium is referred to as EM1. In some embodiments, EM1 additionally
comprises a prostaglandin pathway activator such as PGE2 and/or AA.
In some embodiments, EMI comprises a TGF-beta inhibitor such as
A83-01. More preferably, the EM1 additionally comprises a
prostaglandin pathway activator and a TGF-beta inhibitor. The
inventors have found that a medium containing Wnt and Noggin is
ideal for stimulating initial expansion of cells. Thus, in some
embodiments, the EM1 medium is used for just 1 passage or 1 week
but it is also envisaged that EM1 medium can be used for around a
year because it is not harmful for the cells. Thus, in some
embodiments, an EM1 medium is used for culturing cells from day 0
to day 10, for example from days 0-7, days 0-6, days 0-5, days 0-4,
days 0-3, days 0-2, days 0-1, wherein day 0 is the day that the
cells are isolated from their tissue of origin and day 1 is the
subsequent day or is used for 1 or more weeks for example 2, 3, 4
or more weeks or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more
months. In some embodiments, the EM1 medium is used only for the
first day or first two days of culture. In some embodiments, EM1
medium is used for 1 or more passages, for example, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25, 30 or more passages, for example, 20-30
passages, 30-35 passages, 32-40 passages or more. In some
embodiments, the EM1 medium is used subsequent to a freezing step
or any other transportation step involving a medium or temperature
change that does not combine with optimal growth. This "EM1" medium
is preferred for expanding liver cells.
[0187] The EM1 medium is supplemented with one or more of the
compounds selected from the group consisting of FGF, HGF,
Nicotinamide, gastrin, B27, N-acetylcystein and N2. In the case of
starting the cultures from a frozen stock or from a single cell,
the EM1 media is preferably supplemented with a ROCK inhibitor.
Y27632 is the preferred ROCK inhibitor for use in the
invention.
[0188] Thus, in one embodiment there is provided a cell culture
medium which comprises or consists of a basal medium for animal or
human cells to which is added: Epidermal Growth Factor, an FGF (for
example, an FGF able to bind to FGFR2 or FGFR4), preferably FGF10
and HGF as mitogenic growth factors, [0189] a prostaglandin pathway
activator, such as PGE2 and/or AA; [0190] a TGF-beta inhibitor;
[0191] gastrin, Nicotinamide, B27, N2 and N-Acetylcysteine, and
preferably; [0192] a BMP inhibitor, preferably Noggin; and [0193] a
Wnt agonist, preferably R-spondin 1 and/or Wnt-3a.
[0194] B27 (Invitrogen), N-Acetylcysteine (Sigma) and N2
(Invitrogen), Gastrin (Sigma) and Nicotinamide (Sigma) are also
added to the medium defined above and are believed to control
proliferation of the cells and assist with DNA stability. In the
context of the invention, Nicotinamide is also referred to herein
as "Nic".
[0195] `N2 Supplement` is available from Invitrogen, Carlsbad,
Calif.; www.invitrogen.com; catalog no. 17502-048; and from PAA
Laboratories GmbH, Pasching, Austria; www.paa.com; catalog no.
F005-004; Bottenstein & Sato, PNAS, 76(1):514-517, 1979. N2
Supplement is supplied by PAA Laboratories GmbH as a 100.times.
liquid concentrate, containing 500 .mu.g/ml human transferrin, 500
.mu.g/ml bovine insulin, 0.63 .mu.g/ml progesterone, 1611 .mu.g/ml
putrescine, and 0.52.mu.g/ml sodium selenite. N2 Supplement may be
added to a culture medium as a concentrate or diluted before
addition to a culture medium. It may be used at a 1.times. final
concentration or at other final concentrations. Use of N2
Supplement is a convenient way to incorporate transferrin, insulin,
progesterone, putrescine and sodium selenite into a culture medium
of the invention. In some embodiments in which the medium comprises
B27, it does not also comprise N2. The embodiments of the present
invention can therefore be adapted to exclude N2 when B27 is
present, if desired.
[0196] B27 Supplement' (available from Invitrogen, Carlsbad,
Calif.; www.invitrogen.com; currently catalog no. 17504-044; and
from PAA Laboratories GmbH, Pasching, Austria; www.paa.com; catalog
no. F01-002; Brewer et al., J Neurosci Res., 35(5):567-76, 1993)
may be used to formulate a culture medium that comprises biotin,
cholesterol, linoleic acid, linolenic acid, progesterone,
putrescine, retinol, retinyl acetate, sodium selenite,
tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin,
insulin and transferrin. B27 Supplement is supplied by PAA
Laboratories GmbH as a liquid 50.times. concentrate, containing
amongst other ingredients biotin, cholesterol, linoleic acid,
linolenic acid, progesterone, putrescine, retinol, retinyl acetate,
sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol
(vitamin E), albumin, insulin and transferrin. Of these ingredients
at least linolenic acid, retinol, retinyl acetate and
tri-iodothyronine (T3) are nuclear hormone receptor agonists. B27
Supplement may be added to a culture medium as a concentrate or
diluted before addition to a culture medium. It may be used at a
1.times. final concentration or at other final concentrations. Use
of B27 Supplement is a convenient way to incorporate biotin,
cholesterol, linoleic acid, linolenic acid, progesterone,
putrescine, retinol, retinyl acetate, sodium selenite,
tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin,
insulin and transferrin into a culture medium of the invention.
[0197] For example, a cell culture medium may comprise or consist
of a basal medium to which is added: EGF and R-spondin 1
supplemented with FGF10, HGF and Nicotinamide; for example, EGF (50
ng/ml) and R-spondin 1 (1 ug/ml) supplemented with FGF10 (100
ng/ml), HGF (25-50 ng/ml), Nicotinamide (1-10 mM), a prostaglandin
pathway activator, such as PGE2 (50 nM) and/or AA (10 ug/ml) and a
TGF-beta inhibitor such as A83-01 (500 nM). In some embodiments the
medium additionally comprises a p38 inhibitor. The inventors have
found that this medium may be used for long-term expansion of
cells. Thus, this cell culture medium is preferred for use as an
EM2 of the invention. The basal medium is preferably supplemented
with B27, N2 and 200 ng/ml N-Acetylcysteine. In some embodiments,
the basal medium is Advanced-DMEM/F12. However, any other suitable
basal medium may be used.
[0198] Another example of a cell culture medium, and method of
using this medium comprises or consists of Advanced-DMEM/F12
preferably supplemented with B27, N2, 200 ng/ml N-Acetylcysteine,
50 ng/ml EGF, 1 .mu.g/ml R-spondin1, 10 nM gastrin, 100 ng/ml
FGF10, 10 mM Nicotinamide, 50 ng/ml HGF, 50% Wnt conditioned media,
a prostaglandin pathway activator, such as PGE2 (50 nM) and/or AA
(10 ug/ml) and a TGF-beta inhibitor such as A83-01 (500 nM) and,
preferably 10-100 ng/ml Noggin. Wnt conditioned media comprises
Advanced DMEM, P/S, B27, N2 and also FCS. 293T cells transfected
with Wnt3A expression plasmid produce Wnt. The whole medium is
taken after a few days (i.e. with secreted Wnt) and used as the Wnt
source.
[0199] The invention therefore provides a cell culture medium,
comprising or consisting of a basal medium for animal or human
cells to which is added: [0200] Epidermal Growth Factor, an FGF
able to bind to FGFR2 or FGFR4, preferably FGF10 and HGF as
mitogenic growth factors, [0201] a prostaglandin pathway activator,
such as PGE2 and/or AA, [0202] a TGF-beta inhibitor; [0203]
gastrin, Nicotinamide, B27, N2 and N-Acetylcystein, and preferably
[0204] a BMP inhibitor more preferably Noggin and [0205] a Wnt
agonist, more preferably R-spondin 1 and/or Wnt-3 a.
[0206] The invention thus encompasses a first preferred culture
medium comprising or consisting of a basal medium for animal or
human cells to which is added:
[0207] Epidermal Growth Factor, FGF10 and HGF as mitogenic growth
factors, [0208] a prostaglandin pathway activator, such as PGE2
and/or AA, [0209] a TGF-beta inhibitor; [0210] gastrin,
Nicotinamide, B27, N2 and N-Acetylcysteine, [0211] a BMP inhibitor
more preferably Noggin and [0212] a Wnt agonist, more preferably
R-spondin 1 and Wnt-3a.
[0213] In some embodiments, a p38 inhibitor is added to the
expansion medium.
[0214] This medium may be used as an EM1 cell culture medium of the
invention to stimulate initial expansion of cells. In some
embodiments, the medium used as an EM1 cell culture medium
comprises all the components of an EM2 culture medium of the
invention and additionally comprises Wnt-3a and Noggin.
[0215] In embodiments in which the basal medium is supplemented
with N-Acetylcysteine, B27 and N2, the following are preferably
added to the culture media: EGF, R-spondin1, gastrin, FGF10,
Nicotinamide and HGF and Wnt-conditioned media and a prostaglandin
pathway activator, such as PGE2 and/or AA. Preferably, the basal
medium is supplemented with N-Acetylcysteine, EGF, R-spondin1,
gastrin, FGF10, Nicotinamide and HGF and Wnt-conditioned media and
a prostaglandin pathway activator, such as PGE2 and/or AA in
accordance with the quantities described hereinabove. Preferably, a
TGF-beta inhibitor is also present at the quantities described
herein.
[0216] For example, in some embodiments the basal medium may be
supplemented with 150 ng/ml to 250 ng/ml N-Acetylcysteine;
preferably, the basal medium is supplemented with, about or exactly
200 ng/ml N-Acetylcysteine. For example, in some embodiments the
basal medium may be supplemented with 40 ng/ml to 60 ng/ml EGF;
preferably, the basal medium is supplemented with about or exactly
50 ng/ml EGF. For example, in some embodiments the basal medium may
be supplemented with 0.5 .mu.g/ml to 1.5 .mu.g/ml R-spondin1;
preferably, the basal medium is supplemented with about or exactly
1 .mu.g/ml R-spondin1. For example, in some embodiments the basal
medium may be supplemented with 5 nM to 15 nM gastrin; preferably,
the basal medium is supplemented with about or exactly 10 nM
gastrin. For example, in some embodiments the basal medium may be
supplemented with 25-200 ng/ml FGF10, for example 70 ng/ml to 130
ng/ml FGF10; preferably, the basal medium is supplemented with
about or exactly 100 ng/ml FGF10. For example, in some embodiments
the basal medium may be supplemented with 5 mM to 15 mM
Nicotinamide; preferably, the basal medium is supplemented with
about or exactly 10 mM Nicotinamide. For example, in some
embodiments the basal medium may be supplemented with 25 ng/ml to
100 ng/ml HGF, for example 35 ng/ml to 65 ng/ml HGF; preferably,
the basal medium is supplemented with about or exactly and 50 ng/ml
HGF. For example, in some embodiments the basal medium may be
supplemented with 35% to 65% Wnt-conditioned media; preferably, the
basal medium is supplemented with about or exactly 50%
Wnt-conditioned media.
[0217] For example, in some embodiments, the liver expansion medium
is supplemented with an activator of the prostaglandin signalling
pathway (see FIG. 24). For example, the liver expansion medium may
be supplemented with any one or more of the compounds selected from
the list comprising: Phospholipids, Arachidonic acid (AA),
prostaglandin E2 (PGE2), prostaglandin G2 (PGG2), prostaglandin F2
(PGF2), prostaglandin H2 (PGH2), prostaglandin D2 (PGD2). For
example, in some embodiments, the liver expansion medium is
supplemented with PGE2 and/or AA. In some embodiments, PGE2 is
added to the liver expansion medium to a final concentration of at
least 10 nM, for example between 10 nM and 500 nM, between 10 nM,
and 400 nM, between 10 nM and 300 nM, between 10 nM and 200 nM,
between 10 nM and 100 nM, between 20 nM and 50 nM. In a preferred
embodiment, PGE2 is added to the liver expansion medium to a final
concentration of 50 nM. In some embodiments, AA is added to the
liver expansion medium to a final concentration of at least 1
ug/ml, for example between 1 ug/ml and 1000 ug/ml, between 1 ug/ml
and 500 ug/ml, between 1 ug/ml and 100 ug/ml, between 1 ug/ml and
50 ug/ml, or between 5 ug/ml and 10 ug/ml. In a preferred
embodiment, AA is added to the medium to a final concentration of
10 ug/ml.
[0218] In some embodiments one or both of gastrin and N2 are not
present in the cell culture medium.
[0219] Preferably, the basal medium is advanced-DMEM/F12.
[0220] This first culture medium (for example, EM1, EM2 or both EM1
and EM2) is preferably used during the first two weeks of the
culture method of the invention. However, it may be used for a
shorter time period, such as for 1, 2, 3, 5, 7, or 10 days, or a
longer time period, such as 3, 4, 5, 10, 20 or more weeks, 5 months
or more, for example, 6, 7, 8, 9, 10, 11, 12 months or more.
Differentiation Medium (DM) for Liver:
[0221] In another aspect, there is provided a second cell culture
medium which comprises or consists of a basal medium for animal or
human cells to which is added: EGF, a TGF-beta inhibitor, a Notch
inhibitor and a prostaglandin pathway activator, such as PGE2
and/or AA. The inventors have found that this medium is useful for
differentiating cells. The medium used for differentiating the
cells may be referred to herein as DM.
[0222] Preferably, the second cell culture medium also comprises
FGF and/or HGF.
[0223] In one embodiment, the second culture medium comprises or
consists of a basal medium for animal or human cells to which is
added: [0224] Epidermal Growth Factor, FGF10 and HGF as mitogenic
growth factors; [0225] a Notch inhibitor; [0226] a TGF-beta
inhibitor; and [0227] a prostaglandin pathway activator, such as
PGE2 and/or AA.
[0228] In one embodiment, the TGF-beta inhibitor is A83-01 and/or
the Notch inhibitor is DAPT. In another embodiment, the DM cell
culture medium additionally comprises Dexamethasone. In another
embodiment, the DM cell culture medium additionally comprises
Oncostatin M. In another embodiment, the DM cell culture medium
additionally comprises gastrin.
[0229] A preferred second cell culture medium, and method of using
this medium, is described in the examples, and comprises or
consists of a basal medium to which is added: 50 ng/ml EGF, 100
ng/ml FGF10, 50 nM A8301 and 10 uM DAPT. Advanced-DMEM/F12 may be
used as the basal medium as may any other suitable basal
medium.
[0230] In some embodiments, the differentiation medium for liver
cells, for example for human liver cells, comprises or consists of
a basal medium (for example comprising Advanced DMEM/F12, B27
(50.times.), n-Acetylcystein (1 mM) glutamin/glutamax), Noggin
(preferably 100 ng/ml), EGF (preferably 50 ng/ml), gastrin
(preferably 10 nM), TGF-beta inhibitor, such as A83-01 (preferably
50 nM) and a gamma-secretase inhibitor (for example DAPT/DBZ)
(preferably 10 uM).
[0231] In some embodiments, the differentiation medium for liver
cells, for example for mouse liver cells, comprises or consists of
a basal medium (for example comprising Advanced DMEM/F12, B27
(50.times.), n-Acetylcystein (preferably 1 mM) glutamin/glutamax),
EGF (preferably 50 ng/ml), FGF10 (preferably 100 ng/ml) gastrin
(preferably 10 nM), TGF-beta inhibitor, such as A83-01 (preferably
50 nM) and a gamma-secretase inhibitor (for example DAPT/DBZ)
(preferably 10 uM).
[0232] In some embodiments, the second cell culture medium does not
comprise R-spondin or Wnt. In some embodiments, the second cell
culture medium does not comprise a Wnt agonist. In some
embodiments, the second cell culture medium does not comprise
Nicotinamide. In some embodiments, the second cell culture medium
does not comprise a BMP inhibitor. In some embodiments, the second
cell culture medium does not comprise a prostaglandin pathway
activator, such as PGE2 and/or AA.
[0233] The inventors have discovered that R-spondin1 and
Nicotinamide both inhibit the expression of the mature hepatocyte
marker CYP3A11 and yet promote the expression of the hepatoblast
marker albumin. Therefore, to increase differentiation of the cells
to more mature liver fates, the inventors removed R-spondin and
Nicotinamide from the cell culture. The inventors have also
discovered that the expression of specific biliary transcription
factors is highly upregulated in expansion cultures containing
R-spondin1, indicating that the culture gene expression was
unbalanced towards a more biliary cell fate. Notch and TGF-beta
signaling pathways have been implicated in biliary cell fate in
vivo. In fact, deletion of Rbpj (essential to achieve active Notch
signalling) results in abnormal tubulogenesis (Zong Y. Development
2009) and the addition of TGF-beta to liver explants facilitates
the biliary differentiation in vitro (Clotman F. Genes and
Development 2005). Since both Notch and TGF-beta signalling
pathways were highly upregulated in the liver cultures (FIG. 22)
the inventors reasoned that inhibition of biliary duct cell-fate
might trigger the differentiation of the cells towards a more
hepatocytic phenotype. It was found that addition of a TGF-beta
inhibitor (such as A8301) and a Notch inhibitor (such as DAPT) to a
differentiation medium that preferably does not contain R-spondin
or Wnt, enhances the expression of mature hepatocyte markers and
increases the number of hepatocyte-like cells (for example, see
example 5).
General Culture Media
[0234] A cell culture medium according to the invention allows the
survival and/or proliferation and/or differentiation of epithelial
stem cells or isolated crypts on an extracellular matrix. In some
embodiments, a cell culture medium according to the invention
allows the survival and/or proliferation and/or differentiation of
an organoid of the invention, such as a crypt-villus organoid, a
colon organoid, a pancreatic organoid, a gastric organoid, a
Barret's Esophagus organoid, an adenocarcinoma organoid or a colon
carcinoma organoid on an extracellular matrix. In some embodiments,
a cell culture medium according to the invention allows the
survival and/or proliferation and/or differentiation of an organoid
of the invention, such as a small intestinal (crypt-villus)
organoid, a colon organoid, a pancreatic organoid, a gastric
organoid, a Barret's Esophagus organoid, an adenocarcinoma
organoid, a carcinoma organoid, a colon carcinoma organoid, a
prostate organoid or a prostate carcinoma organoid on an
extracellular matrix. Preferably, in embodiments in which a
TGF-beta inhibitor and/or p38 inhibitor is present the cell culture
medium allows the survival and/or proliferation, preferably the
survival and proliferation of a population of cells or organoid of
the invention. Preferably, embodiments in which a TGF-beta
inhibitor and/or p38 inhibitor is initially present in a cell
culture medium but is then removed from the medium (e.g. by failing
to add it when the medium is refreshed), allow the survival and/or
differentiation, preferably the survival and differentiation of a
population of cells or organoid of the invention.
[0235] In some embodiments, a p38 inhibitor is added to any of the
media described herein.
[0236] The term cell culture medium is synonymous with medium,
culture medium or cell medium.
Uses of Culture Media of the Invention
[0237] The invention also provides the use of a culture medium of
the invention for expanding and/or differentiating a stem cell,
population of stem cells, tissue fragment or organoid.
[0238] In some embodiments, the stem cell, population of stem
cells, tissue fragment or organoid is selected from the group
consisting of one or more intestinal stem cells, small intestinal
crypts, colonic crypts, gastric stem cells, liver stem cells,
pancreas stem cells and prostate stem cells.
[0239] In some embodiments, the stem cell, population of stem
cells, tissue fragment or organoid is obtainable from a normal
tissue.
[0240] In some embodiments, the stem cell, population of stem
cells, tissue fragment or organoid is obtainable from a diseased
tissue, for example from an adenoma, a carcinoma, an
adenocarcinoma, an intestine of a patient having cystic fibrosis or
an intestine of a patient having inflammatory bowel disease.
Stem Cells Cultured According to the Invention
[0241] Stem cells are found in many organs of adult humans and
mice. Although there may be great variation in the exact
characteristics of adult stem cells in individual tissues, adult
stem cells share at least the following characteristics: they
retain an undifferentiated phenotype; their offspring can
differentiate towards all lineages present in the pertinent tissue;
they retain self-maintenance capabilities throughout life; and they
are able to regenerate the pertinent tissue after injury. Stem
cells reside in a specialised location, the stem cell niche, which
supplies the appropriate cell-cell contacts and signals for
maintenance of said stem cell population. The stem cells according
to the invention preferably express Lgr5.
[0242] In one embodiment, the invention provides a population of
cells or one or more organoids comprising said stem cells that have
been generated or obtained by culturing stem cells or tissue
fragments according to the invention, which have been cultured for
at least 3 months, preferably at least 4 months, at least 5 months,
at least 6 months, at least 7 months, at least 9 months, or at
least 12 months or more.
[0243] A `population` of cells is any number of cells greater than
1, but is preferably at least 1.times.10.sup.3 cells, at least
1.times.10.sup.4 cells, at least 1.times.10.sup.5 cells, at least
1.times.10.sup.6 cells, at least 1.times.10.sup.7 cells, at least
1.times.10.sup.8 cells, or at least 1.times.10.sup.9 cells.
[0244] The stem cells of the invention cultured according to the
invention may be human stem cells. The stem cells of the invention
cultured according to the invention may be epithelial stem
cells.
[0245] In some embodiments, the stem cells of the invention and/or
cultured according to the invention are not embryonic stem cells.
In some embodiments the stem cells of the invention and/or cultured
according to the invention are not human embryonic stem cells.
Preferably, the stem cells of the invention are adult stem
cells.
[0246] In a preferred embodiment, the stem cells may be human
epithelial stem cells. Human epithelial stem cells include stem
cells of human epithelial tissue origin. These include, but are not
limited to pancreatic, small intestinal, large intestinal, corneal,
olfactory, respiratory tissues, gastric tissues, liver and skin,
mammary and/or prostatic tissues, for example, a tissue selected
from the group consisting of pancreatic, small intestinal, large
intestinal, corneal, olfactory, and respiratory tissues. Epithelial
stem cells are able to form the distinct cell types of which the
epithelium is composed. Some epithelia, such as skin or intestine,
show rapid cell turnover, indicating that the residing stem cells
must be continuously proliferating. Other epithelia, such as the
liver or pancreas, show a very slow turnover under normal
conditions.
Intestinal Stem Cells
[0247] The self-renewing epithelium of the small intestine is
ordered into crypts and villi (Gregoreff and Clevers, 2005 Genes
Dev 19, 877-90). Each cell along the crypt-villus axis is
polarized, whereby cells on the top of the intestinal villi, or in
the upper positions of colonic crypts, are the most differentiated
and are continuously lost into the lumen by apoptosis. Continuous
proliferation of stem cells residing in the base of the crypts, and
massive proliferation of progenitor cells residing in the middle of
the crypts, ensures proper replacement of the shed cells. There is
a resulting epithelial turnover time of 5 days in the mouse.
Self-renewing stem cells have long been known to reside near the
crypt bottom and to produce the rapidly proliferating transit
amplifying (TA) cells capable of differentiating towards all
lineages. The estimated number of stem cells is between 4 and 6 per
crypt (Bjerknes and Cheng, 1999 Gastroenterology 1 16, 7-14). Three
differentiated cell types, enterocytes, goblet cells and
enteroendocrine cells, form from TA cells and continue their
migration in coherent bands along the crypt-villus axis. Each
villus receives cells from multiple different crypts. The fourth
major differentiated cell-type, the Paneth cell, resides at the
crypt bottom.
[0248] The colon resembles the small intestine but with a flat
surface epithelium rather than villi. Colon crypts are organized
like small intestinal crypts. Paneth cells are not present in colon
crypts; instead there are so-called "Deep Crypt Secretory" cells.
The flat surface of the epithelium contains the differentiated
cells (colonocytes and secretory cells). Differentiated goblet
cells occur throughout the crypt, also intermingled with transit
amplifying cells.
Isolation of Tissue Fragments and Stem Cells
[0249] Crypts can be isolated from the small and large intestine,
including the duodenum, jejunum, ileum and colon, and the pyloric
and corpus region of the stomach by protocols that are known to the
skilled person. For example, crypts can be isolated by incubation
of isolated tissue with chelating agents that release cells from
their calcium- and magnesium-dependent interactions with the
basement membrane and stromal cell types. After washing the tissue,
the epithelial cell layer is scraped from the submucosa with a
glass slide and minced. This is followed by incubation in trypsin
or, more preferred, EDTA and/or EGTA and separation of undigested
tissue fragments and single cells from crypts using, for example,
filtration and/or centrifugations steps. Other proteolytic enzymes,
such as collagenase and/or dispase I, can be used instead of
trypsin. Similar methods are used to isolate fragments of the
pancreas and stomach. Similar methods may be used to isolated
fragments of other tissues described herein. The culture media of
the invention are suitable for culturing such tissue fragments (see
Example 1).
[0250] A culture medium according to the invention allows the
establishment of long-term culture conditions under which single
crypts undergo multiple crypt fission events, while simultaneously
generating villus-like epithelial domains in which all
differentiated cell types are present. Cultured crypts undergo
dramatic morphological changes after taking them into culture. The
upper opening of freshly isolated crypts becomes sealed and this
region gradually balloons out and becomes filled with apoptotic
cells, much like apoptotic cells are pinched off at the villus tip.
The crypt region undergoes continuous budding events which create
additional crypts, a process reminiscent of crypt fission. In a
preferred embodiment of the invention, the organoids comprise
crypt-like extensions which comprise all differentiated epithelial
cell types, including proliferative cells, Paneth cells,
enterocytes and goblet cells. No myofibroblasts or other
non-epithelial cells were identified in the organoids at any
stage.
[0251] Expansion of the budding crypt structures creates organoids,
comprising crypt-like structures surrounding a central lumen lined
by a villus-like epithelium and filled with apoptotic cell bodies.
The crypt-villus organoids comprise a central lumen lined by a
villus-like epithelium. The lumen is opened at consecutive time
intervals to release the content into the medium.
[0252] A similar crypt-villus organoid structure is formed when
single epithelial stem cells are cultured. After about one week,
structures are formed that strongly resemble the crypt-villus
organoid structures that are obtained with intact crypts.
[0253] Methods to isolate stem cells are known and suitable methods
for use with this invention can be selected by the skilled person
depending on the stem cell type that is used. For example,
isolation of epithelial stem cells may be performed using compounds
that bind to Lgr5 and/or Lgr6, which are unique cell surface
markers on epithelial stem cells. Examples of such compounds are
anti-Lgr5 and anti-Lgr6 antibodies.
[0254] In some embodiments of the invention, single Lgr5+
epithelial stem cells, for example from the colon, small intestine,
or pancreas, may be used to form organoids, such as colonic,
crypt-villus or pancreatic organoids respectively.
[0255] In a further example, single Lgr5+ epithelial stem cells
from the liver, prostate or stomach may be used to obtain
organoids, such as liver, prostate or gastric organoids
respectively.
[0256] In an alternative embodiment, tissue fragments, such as
cultured crypts from the intestinal tract, comprising Lgr5+ stem
cells may be used to obtain organoids using methods and culture
media described herein.
[0257] In some embodiments the single Lgr5+ epithelial stem cell or
tissue fragment may be a cancer stem cell or cancer tissue
fragment, for example from a carcinoma or adenocarcinoma. In some
embodiments the single Lgr5+ epithelial stem cell may be a stem
cell or tissue fragment from a neoplastic pathology or diseased
tissue, for example Barrett's esophagus, cystic fibrosis or
adenoma. Organoids obtained from cancerous, neoplastic or diseased
starting material have characterisitics resembling the in vivo
starting material and therefore are useful as a research tool for
drug screening, target validation, target discovery, toxicology and
toxicology screens, personalized medicine, regenerative medicine
and ex vivo cell/organ models, for example disease models. In one
embodiment, the invention provides organoids generated or obtained
by culturing human stem cells or tissue fragments according to a
method of the invention. In one embodiment, the invention provides
crypt-villus organoids or gastric organoids or pancreatic organoids
or colon organoids or Barrett's Esophagus organoids or
adenocarcinoma organoids or colon carcinoma organoids generated or
obtained by culturing human stem cells or tissue fragments
according to a method of the invention. In one embodiment, the
invention provides prostate organoids generated or obtained by
culturing human stem cells or tissue fragments according to a
method of the invention. Such a population of organoids, for
example, crypt-villus, gastric or pancreatic organoids, generated
or obtained by culturing human stem cells or tissue fragments
according to a method of the invention, may each comprise more than
10, preferably more than 20, more preferably more than 40
organoids. Said collection of organoids preferably comprises at
least 10% viable cells, more preferred at least 20% viable cells,
more preferred at least 50% viable cells, more preferred at least
60% viable cells, more preferred at least 70% viable cells, more
preferred at least 80% viable cells, more preferred at least 90%
viable cells. Viability of cells may be assessed using Hoechst
staining or Propidium Iodide staining in FACS.
[0258] The inventors have shown that the culture media and methods
of the invention may be used for culture of cancer cell lines,
including colorectal cancer and adenocarcinoma (see Example 1). As
explained in Example 1, the culture technology is widely applicable
as a research tool for infectious, inflammatory and neoplastic
pathologies. Accordingly, the stem cells according to the invention
may be cancer stem cells. In some embodiments of the invention,
cancer stem cells can form adenoma or colon cancer organoids. In
some embodiments, these organoids comprise cells which are
Ki67+(Thermo Scientific* Cellomics, Millipore).
[0259] Similarly, the inventors have shown that the culture media
and methods of the invention may be used for culturing stem cells
with other diseased genotypes and/or phenotypes. For example,
intestinal stem cells taken from patients with cystic fibrosis can
be expanded using the culture media and methods of the invention.
These stem cells maintain the cystic fibrosis genotype and
phenotype. Therefore, in some embodiments of the invention, the
stem cells are taken from a patient with a disease, for example
cystic fibrosis, inflammatory bowel disease (such as Crohn's
disease), carcinoma, adenoma, adenocarcinoma, colon cancer,
diabetes (such as type I or type II), Barrett's esophagus Gaucher's
disease, alpha-1-antitrypsin deficiency, Lesch-Nyhan syndrome,
anaemia, Schwachman-Bodian-Diamond syndrome, polycythaemia vera,
primary myelofibrosis, glycogen storage disease, familial
hypercholestrolaemia, Crigler-Najjar syndrome, hereditary
tyrosinanaemia, Pompe disease, progressive familial cholestasis,
Hreler syndrome, SCID or leaky SCID, Omenn syndrome, Cartilage-hair
hypoplasia, Herpes simplex encephalitis, Scleroderma, Osteogenesis
imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy,
Dyskeratosis congenitor, etc. In some embodiments of the invention,
disease organoids can be obtained by culturing stem cells taken
from a human or animal with a disease. Disease organoids still have
characteristics of the tissue from which they were obtained.
Therefore, a cystic fibrosis small intestinal organoid grown from a
small intestinal crypt falls within the definition of a small
intestinal organoid. Similarly, a colon carcinoma organoid falls
within the definition of a colon organoid.
[0260] There is some confusion in the literature as to the
definition of a cancer stem cell. Here, we follow the consensus
reached at a recent AACR workshop (Clarke et al., 2006. Cancer Res.
66:9339-44), which states that the cancer stem cell "is a cell
within a tumor that possesses the capacity to self-renew and to
cause the heterogeneous lineages of cancer cells that comprise the
tumor. Cancer stem cells can thus only be defined experimentally by
their ability to recapitulate the generation of a continuously
growing tumor". Alternative terms in the literature include
tumor-initiating cell and tumorigenic cell. Assays for cancer stem
cell activity need to address the potential of self-renewal and of
tumor propagation. The gold-standard assay currently is serial
xeno-transplantation into immunodeficient mice. In addition, cancer
stem cells in the context of this invention normally express Lgr5.
However, in some embodiments, cancer initiating/propagating/stem
cells that do not express Lgr5 can also be cultured by the culture
media and methods of the invention.
Genomic and Phenotypic Integrity of Stem Cells and Organoids
Comprising Said Stem Cells
[0261] Clinical and research applications for stem cells and their
differentiated progeny require reproducible stem cell culture
methods that provide populations of cells of suitable quality.
Generally, in vitro expansion of stem cells aims to provide a
population of cells which resemble their in vivo counterparts as
closely as possible. This property is herein referred to as the
"genomic and phenotypic integrity" of the cells. Organoids obtained
by culturing diseased cells, such as cancer cells or cystic
fibrosis cells, also resemble their in vivo counterparts i.e. they
maintain their disease genotype and/or phenotype and therefore,
also maintain their "genomic and phenotypic integrity" in that
sense i.e. they maintain the genetic or phenotypic instability
characteristic of the disease that is remincent of the in vivo
situation. Therefore, in some embodiments, the invention provides
"normal" organoids obtained from healthy tissue. In other
embodiments, the invention provides "disease" organoids, such as
cancer organoids (for example, colon carcinoma organoids or
adenocarcinoma organoids) or cystic fibrosis small intestinal
organoids obtained from diseased tissue.
[0262] For the first time, the inventors have discovered that it is
possible to expand human epithelial stem cells in culture, with
minimal loss of genomic and phenotypic integrity, for at least 3
months, preferably at least 4 months, at least 5 months, at least 6
months, at least 7 months, at least 9 months, or at least 12 months
or more (see Example 1). Under the improved culture conditions of
the invention, human intestinal organoids displayed budding
organoid structures, rather than the cystic structures seen under
previous culture conditions. Metaphase spreads of organoids more
than 3 months old consistently revealed 46 chromosomes in each of
the 20 cells taken from three different donors. Furthermore,
microarray analysis revealed that the stem cells in culture
possessed similar molecular signatures to intestinal crypt cells
including intestinal stem cell genes.
[0263] Therefore, in some embodiments the invention provides
organoids that have been grown for at least 3 months, preferably at
least 4 months, at least 5 months, at least 6 months, at least 7
months, at least 9 months, or at least 12 months or more with
minimal loss of genomic and phenotypic integrity.
[0264] In some embodiments, the invention provides human intestinal
organoids comprising budding structures. In some embodiments of the
invention, human intestinal organoids do not comprise cystic
structures. In some embodiments of the invention, human intestinal
organoids comprise more budding structures than cystic structures.
The inventors also demonstrated that the human intestinal organoids
generated by media and methods of the present invention, mimicked
in vivo cell fate decisions in response to external factors. For
example, it has previously been shown that Notch inhibition in
intestinal stem cells, terminates intestinal epithelial
proliferation and induces goblet cell hyperplasia in vivo. The
inventors were able to show that the intestinal organoids of the
invention, when treated with a Notch inhibitor, ceased
proliferation and most cells converted into goblet cells within 3
days.
[0265] These results show the dramatic improvement in the genomic
and phenotypic integrity of the stem cells and organoids produced
by the methods and media of the present invention compared to
previous methods and media.
[0266] The genomic integrity of stem cells of the invention can be
confirmed by karyotype analysis. Stem cells and their progeny can
be karyotyped using known methods as described in Sato, T et al.,
Single Lgr5 stem cells build crypt-villus structures in vitro
without a mesenchymal niche. Nature 459, 262-265, 2009.
[0267] A "normal karyotype" is one where all chromosomes are
present (i.e. euploidy) with no noticeable alterations.
Accordingly, in preferred embodiments of the invention more than
50%; more than 70%; more than 80%; more than 90%; more than 95%; or
more than 99% of the stem cells and differentiated cells in an
expanded population exhibit normal karyotypes after 1, 2, 3, 4, 5,
6, 9, 12 or more months. The term "expanded population" encompasses
organoids.
[0268] A "normal phenotype" refers to cells which display, to a
first approximation, the same visual characteristics, gene
expression and behaviour as the average in vivo counterpart cell.
In preferred embodiments of the invention more than 50%; more than
70%; more than 80%; more than 90%; more than 95%; or more than 99%
of the stem cells in an expanded population cultured according to
the invention exhibit normal phenotypes after 1, 2, 3, 4, 5, 6, 9,
12 or more months.
[0269] For example, visually a normal phenotype may be judged by
the number of dead cells outside the organoid, the amount of
`budding` of the organoid compared to cystic growth (budding
structures are preferred), and the overall integrity of the single
layer of epithelial cells (e.g. columnar squamous phenotype). In
addition the cell types present may help to judge whether an
organoid is visually "normal".
[0270] Preferred properties of the stem cells and organoids of the
invention are outlined below.
Stem Cell Markers
[0271] When mouse genes are referred to herein, a human organoid of
the invention may have a similar gene profile but wherein the human
gene counterparts are substituted for the mouse genes. Thus, also
provided by the invention is a human organoid having a gene
expression profile as described herein, but in respect of the
corresponding human genes. The human counterparts of the mouse
genes listed herein will be readily available to the skilled
person.
[0272] In one embodiment, the invention provides a population of
adult stem cells characterised by natural expression of Lgr5. In a
preferred embodiment, the invention provides a population of adult
stem cells characterised by natural expression of at least Lgr5 and
one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22 or 23) of stem cell markers from the
group consisting of: LGR4, epcam, Cd24a, Cdca7, Axin, CK19, Nestin,
Somatostatin, CXCR4.sup.+, CD133.sup.+, DCAMKL-1, CD44, Sord, Sox9,
CD44, Prss23, Sp5, Hnf1.alpha., Hnf4a, Sox9, KRT7 and KRT19,
Tnfrsf19. The stem cell markers may be tissue specific. For
example, pancreatic stem cells or organoids may be characterised by
natural expression of one or more (for example 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 1314, or 15 for example, 1, 2, 3 or 4) of: CK19,
Nestin, Somatostatin, insulin, glucagon, CXCR4.sup.+, Ngn3, Pdx1,
NeuroD, Nkx2.2, Nkx6.1, Pax6, Mafa, Hnf1b, optionally Tnfrsf19 at a
significant level; gastric organoids may be characterised by
natural expression of one or more (for example 1, 2, 3 or 4) of:
CD133.sup.+, DCAMKL-1, CD44, optionally Tnfrsf19 at a significant
level; and crypt-villus organoids may be characterised by
expression of one or more or all (for example 1 or 2) of: Sord
and/or Prss23, at a significant level or all genes of table/FIG.
14, for example, at a significant level.
[0273] The term "significant level" as used herein in the context
of marker expression is used synonymously with the term "detectable
level", as described below.
[0274] Small intestinal and gastric organoid cell populations also
express markers of progenitor populations common to the small
intestine and stomach, such as one or both of Cd44 and Sox9 (Barker
& Huch et al Cell stem cell 2010). These are highly expressed
in the stem cells according to the invention. Cells according to
this aspect of the invention may also up-regulate Wnt target genes,
including for example, one, two or all of MMP7, Sp5 Tnfrs 19 and
axin2. This provides strong evidence of the requirement for an
active and robust canonical Wnt signalling activity to maintain the
self renewing capacity of these cultures.
[0275] The inventors have observed that expression of the `stem
cell` genes is present in the early organoids at a level
significantly higher then the differentiated cells that become the
offspring of these stem cells. For example, the genes LGR5, LGR4,
Epcam, CD44, Tnfrsf19, Sox9, Cd24a, Sp5, Prom1/CD133, Cdca7, are
preferably expressed in the organoids of the invention but are
preferably significantly downregulated upon differentiation of the
pancreas, liver, small intestine and colon organoids. In addition,
the genes RNF43 and ZNRF3 are preferably expressed in the organoids
of the invention.
[0276] By "natural expression" is meant that the cells have not
been manipulated recombinantly in any way, i.e., the cells have not
been artificially induced to express these markers or to modulate
these markers' expression by introduction of exogenous genetic
material, such as introduction of heterologous (non-natural) or
stronger promoters or other regulatory sequences operably linked to
either the endogenous genes or exogenously-introduced forms of the
genes. Natural expression is from genomic DNA within the cells,
including introns between the exon coding sequences where these
exist. Natural expression is not from cDNA. Natural expression can
if necessary be proven by any one of various methods, such as
sequencing out from within the reading frame of the gene to check
that no extraneous heterogenous sequence is present. "Adult" means
post-embryonic. With respect to the stem cells of the present
invention, the term "adult stem cell" means that the stem cell is
isolated from a tissue or organ of an animal at a stage of growth
later than the embryonic stage.
[0277] This stem cell population can also be characterised by a
lack of natural expression of certain markers at any significant
level, many of which are associated with cellular differentiation.
Specifically, the cells of the isolated adult stem cell population
do not naturally express one or more of Cd11b, CD13, CD14, AFP,
Pdx1, any CYP member (e.g. CYP3A11, CYP 11A1) at a significant
level. As defined herein, these markers are said be to be negative
markers.
Detecting Markers and Isolating Cells
[0278] The term "expressed" is used to describe the presence of a
marker within a cell. In order to be considered as being expressed,
a marker must be present at a detectable level. By "detectable
level" is meant that the marker can be detected using one of the
standard laboratory methodologies such as PCR, blotting or FACS
analysis. A gene is considered to be expressed by a cell of the
population of the invention if expression can be reasonably
detected after 30 PCR cycles, which corresponds to an expression
level in the cell of at least about 100 copies per cell. The terms
"express" and "expression" have corresponding meanings. At an
expression level below this threshold, a marker is considered not
to be expressed. The comparison between the expression level of a
marker in a cell of the invention, and the expression level of the
same marker in another cell, such as for example an embryonic stem
cell, may preferably be conducted by comparing the two cell types
that have been isolated from the same species. Preferably this
species is a mammal, and more preferably this species is human.
Such comparison may conveniently be conducted using a reverse
transcriptase polymerase chain reaction (RT-PCR) experiment.
[0279] In some embodiments, a population of cells or an organoid of
the invention is considered to express a marker if at least about
5%, (for example, at least 10%, at least 20%, at least 30%, at
least 40%, at least 60%, at least 70%, at least 80%, at least 90%,
at least 95%, at least 97%, at least 98%, at least 99% or 100%) of
the cells in the cell population or organoid according to the
invention show expression of the marker.
[0280] In some embodiments, the cells express a cell marker at a
significant level if they comprise between 1.times.10.sup.2 to
1.times.10.sup.5, for example 5.times.10.sup.2 to 1.times.10.sup.4
or 1.times.10.sup.3 to 1.times.10.sup.4 fold more copies of the
mRNA encoding the cell marker relative to the number of mRNA copies
of the housekeeping gene GADPH.
[0281] In some embodiments, the expression of a gene in an organoid
or cell of the invention when cultured in expansion medium is
several fold (e.g. at least 1.5 fold, 2 fold, 3 fold, 4 fold, 5
fold) higher than when the organoid or cell is cultured in
differentiation medium or in the fully differentiated adult tissue.
In some embodiments, a cell or organoid of the invention when
cultured under differentiation conditions, exhibits an increase in
expression of genes that are known as differentiation genes
compared to a cell or organoid of the invention when cultured under
expansion conditions and also may show a decrease in the level of
expression of at least one or more stem cell/progenitor genes
compared to a cell or organoid of the invention when cultured in
expansion medium.
[0282] Any one of a number of physical methods of separation known
in the art may be used to select the cells of this aspect of the
invention and distinguish these from other cell types. Such
physical methods may involve FACS and various immuno-affinity
methods based upon makers specifically expressed by the cells of
the invention. As described above, Lgr5, CD44 and Sox9 are three of
the cell markers expressed at high levels in the stem cells of the
invention. Therefore, by way of illustration only, the stem cells
of the invention may be isolated by a number of physical methods of
separation, which rely on the presence of these.
[0283] In one embodiment, the cells of the invention may be
isolated by FACS utilizing an antibody, for example, against one of
these markers. Fluorescent activated cell sorting (FACS) can be
used to detect markers characteristic of a particular cell type or
lineage. As will be apparent to one skilled in the art, this may be
achieved through a fluorescent labeled antibody, or through a
fluorescent labeled secondary antibody with binding specificity for
the primary antibody.
[0284] Examples of suitable fluorescent labels includes, but is not
limited to, FITC, Alexa Fluor.RTM. 488, GFP, CFSE, CFDA-SE, DyLight
488, PE, PerCP, PE-Alexa Fluor.RTM. 700, PE-Cy5 (TRI-COLOR.RTM.),
PE-Cy5.5, PI, PE-Alexa Fluor.RTM. 750, and PE-Cy7. This list is
provided by way of example only, and is not intended to be
limiting.
[0285] It will be apparent to a person skilled in the art that FACS
analysis using an anti-Lgr5 antibody will provide a purified stem
cell population. However, in some embodiments, it may be preferable
to purify the cell population further by performing a further round
of FACS analysis using one or more of the other identifiable
markers.
[0286] Immunohistochemistry may also be used to understand the
distribution and localisation of biomarkers and differentially
expressed proteins in different parts of a cell population or
organoid. Visualising an antibody-antigen interaction can be
accomplished in a number of ways that are well known in the art,
such as those that are described in described in Barker et al,
Identification of stem cells in small intestine and colon by marker
gene Lgr5. Nature, 2007 Oct. 25; 449(7165):1003-7.
[0287] In another embodiment, the cells of the invention may be
isolated by immuno-affinity purification, which is a separation
method well known in the art. By way of illustration only, the
cells of the invention may be isolated by immuno-affinity
purification directed towards c-kit. As will be apparent to one
skilled in the art, this method relies upon the immobilisation of
antibodies on a purification column. The cell sample is then loaded
onto the column, allowing the appropriate cells to be bound by the
antibodies, and therefore bound to the column. Following a washing
step, the cells are eluted from the column using a competitor which
binds preferentially to the immobilised anti-c-kit antibody, and
permits the cells to be released from the column. It will be
apparent to a person skilled in the art that immuno-affinity
purification using an immobilised antibody will provide a purified
cell population. However, in some embodiments, it may be preferable
to purify the cell population further by performing a further round
of immuno-affinity purification using one or more of the other
identifiable markers, and use an aliquot of the isolated clones to
ascertain the expression of other relevant intracellular
markers.
[0288] It will be apparent to a person skilled in the art that LGR5
or stem cell purification can be preceded by any number of
purification steps, such as purification of the epithelium with
methods known in the art, for example EDTA purification or Epcam
FACS sorting of the epithelium.
[0289] It will be apparent to a person skilled in the art that the
sequential purification steps are not necessarily required to
involve the same physical method of separation. Therefore, it will
be clear that, for example, the cells may be purified through a
FACS step using an anti-Lgr5 antibody, followed by an
immuno-affinity purification step using a SSEA-1 affinity column.
In certain embodiments, the cells may be cultured after isolation
for at least about 15, at least about 20 days, at least about 25
days, or at least about 30 days. In certain aspects, the cells are
expanded in culture longer to improve the homogeneity of the cell
phenotype in the cell population.
[0290] Mircroarray analysis, cluster analysis and comparative gene
expression profiling can be used to compare population phenotype
with phenotype of the original parent cells or of the appropriate
in vivo counterparts (Sato T et al., Paneth cells constitute the
niche for Lgr5 stem cells in intestinal crypts. Nature 469
415-418).
[0291] Lineage tracing of Lgr5+ stem cells shows preservation of
crypt-villus characteristics in organoids.
[0292] In another embodiment, high content analysis may be used to
assess phenotypic integrity of stem cells of the invention. For
example, a number of high content screening kits and platforms
exist, such as point scanning 4 color ImageXpress ULTRA (Molecular
Devices, Union City, USA), the spinning disk (nipkow disk) Pathway
855 and 435 from BD Biosciences (formerly Atto Biosciences,
Rockville, Md.), Opera (PerkinElmer Inc., Waltham, Mass.) and the
slit scanning IN Cell 3000 (GE/Amersham Biosciences, Cardiff, UK),
Arrayscan VTI (Cellomics (Cellomics)), IN Cell Analyzer 2000 (GE
Healthcare Piscataway, N.J., USA), Acumen eX3 (TTP LabTech Ltd
(Acumen eX3)), Scanalyzer (Scanalyzer LemnaTec, Aachen Germany) and
ImageXpress MICRO (Molecular Devices, Union City, USA), IN Cell
1000 (GE/Amersham Biosciences Piscataway, N.J., USA), the Pathway
HT (Becton Dickinson Biosciences) and the ImageXpress MICRO
(Molecular Devices, Union City, USA), Scan R (Olympus Soft Imaging
Solutions, Germany).
Plating Density
[0293] In some embodiments of the invention, single-cell
suspensions or small clusters of cells (2-50 cells/cluster) will
normally be seeded, rather than large clusters of cells, as is
known in the art. As they divide, such cells will be seeded onto a
support at a density that promotes cell proliferation. Typically,
when single cells are isolated the plating density of at least
1-500 cells/well is used, the surface of the well being 0.32
cm.sup.2. When clusters are seeded the plating density is
preferably 250-2500 cells/cm.sup.2. For replating, a density of
between about 2500 cells/cm.sup.2 and about 5,000 cells/cm.sup.2
may be used. During replating, single-cell suspensions or small
cluster of cells will normally be seeded, rather than large
clusters of cells, as in known in the art.
Further Differentiation
[0294] In some embodiments of the invention, certain components of
the expansion medium can be withdrawn to change the cell fate of
the cultured cells towards differentiation. Any components of the
culture medium that are responsible for maintaining an
undifferentiated state and/or activating stem cell or progenitor
genetic programs may be withdrawn from the culture medium.
[0295] In some embodiments of the invention, withdrawal of the
inhibitors of the invention can enable cells of the organoid to
differentiate to mature cells, such as mature goblet and
enteroendocrine cells in crypt-villus organoids. Thus in some
embodiments, the invention provides a method for further
differentiating the organoids using a second culture medium which
does not comprise an inhibitor of the invention. For example, see
Example 1.
[0296] For example, in some embodiments, the inhibitor of TGF-beta
and/or the inhibitor of p38 are withdrawn from the cell culture
medium to allow the cells to differentiate. By "withdrawn" or
"withdrawal" of a component from the cell culture medium is meant
that when the cells are replated and the medium is changed, the
component is not added to the fresh medium.
[0297] In some embodiments, Wnt is present in the expansion medium
but not in the differentiation medium. For example, some
embodiments comprise withdrawal of Wnt for differentiation of colon
organoids to mature enterocytes. Wnt may also be withdrawn to
enable differentiation of crypt-villus organoids.
[0298] In some embodiments, Rspondin is present in the expansion
medium but not in the differentiation medium. For example, some
embodiments comprise withdrawal of Rspondin for differentiation of
colon organoids to mature enterocytes. Rspondin may also be
withdrawn to enable differentiation of crypt-villus organoids. In
some embodiments Rspondin and Wnt may withdrawn to enable
differentiation of crypt-villus organoids.
[0299] In some embodiments, nicotinamide is present in the
expansion medium but not in the differentiation medium. Thus, in
some embodiments, nicotinamide and SB202190 (or another p38
inhibitor) are withdrawn from the cell culture medium to enable
differentiation of the cells, for example, into crypt-villus
organoids or colon organoids.
[0300] Thus, a method of obtaining differentiated cells or
organoids may comprise culturing epithelial cells in a culture
method of the invention which comprises a TGF-beta and/or p38
inhibitor to enable the cells to survive and/or proliferate (i.e.
expansion medium) and then continuing to culture the cell and
replenish the media, wherein the replenished media does not
comprise a TGF-beta inhibitor and/or p38 inhibitor (i.e.
differentiation medium).
[0301] In some embodiments, the differentiation medium comprises
additional components. For example in some embodiments the
differentiation medium comprises a gamma secretase inhibitor, for
example DAPT or DBZ. In some embodiments the differentiation medium
comprises RANK ligand (also referred to herein as RANKL). As
mentioned above, the addition of a gamma secretase inhibitor can
direct the differentiation of intestinal organoids cells, such as
small intestinal organoid cells, towards secretory cells, such as
goblet cells. The addition of RANKL to the culture medium can
direct differentiation intestinal organoid cells such as small
intestinal organoid cells, towards M cells.
[0302] In some embodiments the invention provides a culture medium
for differentiating stem cells from a tissue of interest, wherein
the culture medium comprises or consists of the components of the
culture medium used for expanding the stem cells from the tissue
type of interest but wherein one or more of the following are
excluded from the medium for differentiating stem cells: Wnt,
Rspondin, BMP inhibitor, TGF-beta inhibitor, receptor tyrosine
kinase ligand, p38 inhibitor and nicotinamide.
[0303] Furthermore, the invention provides a method for expanding a
single stem cell or a population of stem cells, preferably to
generate an organoid, wherein the method comprises culturing the
single stem cell or population of stem cells in a culture medium
according to the invention, wherein the method comprises: [0304]
culturing the stem cell, population of stem cells or tissue
fragments in a first expansion medium; [0305] continuing to culture
the stem cell, population of stem cells or tissue fragments and
replenishing the medium with a differentiation medium, wherein the
differentiation medium does not comprise one or more of, preferably
all of the factors selected from: a TGF-beta inhibitor, a p38
inhibitor, nicotinamide and Wnt.
[0306] In general, where a component is described as being
"removed" from a medium, it is meant that that component is not
added when the medium is replenished i.e. the component is excluded
from the replenished medium. When the medium is "replenished" this
can mean that the medium is physically removed from the
extracellular matrix and then replaced with fresh medium.
[0307] For the colon, liver and pancreas, very few differentiated
cells are present in the expansion medium. Only once the expansion
medium is replaced with a differentiation medium, do the cells
begin to differentiate. At this stage, the organoids also start to
lose their stem cells. Differentiated organoids may be appropriate
for certain uses, such as (but not limited to) transplantation,
drug screening of metabolic diseases, toxicology (for example using
liver organoids comprising hepatocytes) and for studying
antibacterial functions of the small intestine. Expanding organoids
may generally be more appropriate for other uses, such as (but no
limited to) regenerative medicine and drug screening, for example
for cancer or cystic fibrosis. Expanding organoids generally have
more growth potential (and thus greater longevity) than
differentiated organoids. In some embodiments, the colon, liver and
pancreatic organoids are not further differentiated.
[0308] The small intestine and prostate organoids differ from the
colon, liver and pancreatic organoids, in that they maintain an
expanding stem cell population whilst also differentiating at the
same time. They do not need to be cultured in a separate
differentiation medium in order for differentiated cell types to be
present. They can be considered to have the properties of both an
expanding and a differentiated organoid. However, to achieve full
differentiation of small intestinal organoids, they can be cultured
in a separate differentiation medium that preferably does not
comprise Wnt3a and that preferably comprises a gamma secretase
inhibitor and/or a RANK ligand (also referred to herein as RANKL).
By "full" differentiation it is meant that all differentiated cell
types are present including goblet cells, neuroendocrine cells,
tuft cells, M-cells, enterocytes and paneth cells. Some of these
differentiated cell types, for example paneth cells, are also
present (sometimes in smaller quantities) in the expanding
organoids.
Organoids
[0309] The cells described above grow into organoids. Accordingly,
an organoid obtainable by a method of the invention is a further
aspect of the invention. Also provided is an organoid as described
herein. The organoid is preferably a human organoid. To the best of
our knowledge, this is the first time that human organoids have
been obtained that are functional and alive after such an extended
period of time (i.e at least 3 months, preferably at least 4
months, at least 5 months, at least 6 months, at least 7 months, at
least 9 months, or at least 12 months or more of culture; see
examples included herein). Functionality is preferably
characterized by the presence of tissue-specific markers and/or by
the structure of said organoid as defined herein. Since the final
amount of organoids obtained correlates with the duration of
culture, the skilled person will understand that the invention is a
pioneer invention and potentially opens new possibilities in for
example regenerative medicine. Thus, there is provided an organoid
as described herein that is functional and alive after at least 3
months (e.g. at least 4, 5, 6, 7, 8 or more months) of culture. For
example, there is provided an organoid as described herein that
retains at least one or more (e.g. 1, 2 or 3) of its structure,
marker expression and function after at least 3 months (e.g. at
least 4, 5, 6, 7, 8 or more months) of culture.
[0310] For example, an organoid according to the present invention
may comprise a population of cells of at least 1.times.10.sup.3
cells, at least 1.times.10.sup.4 cells, at least 1.times.10.sup.5
cells, at least 1.times.10.sup.6 cells, at least 1.times.10.sup.7
cells or more. Each organoid comprises between approximately
1.times.10.sup.3 cells and 5.times.10.sup.3 cells. The inventors
have shown that it is possible to grow organoids from single Lgr5+
stem cells into organoids comprising a population of cells as
described above or comprising a population of cells of
approximately 10.sup.4 cells. For example, it has now been shown
for mouse that it is possible to start growth of an organoid from
single stem cells. Thus, the invention provides a method for
generating an organoid from a single stem cell. In some
embodiments, the organoid comprises approximately 10.sup.4 cells.
In some embodiments, 10-20, or 20-30 or 30-40 or 40-50 organoids
may be grown together in one well of a 24 well plate.
[0311] In some embodiments, the invention provides an organoid or
population of cells, which is capable of surviving in culture for
at least 3 months, for example at least 4 months, at least 5
months, at least 6 months, at least 7 months, at least 9 months, or
at least 12 months or more, when cultured in a culture medium of
the invention.
[0312] In some embodiments, the invention provides an organoid or
population of cells, wherein the organoid or population of cells
expands at a rate of at least 3 fold, at least 4 fold, at least 5
fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9
fold or at least 10 fold per week.
[0313] Preferably the population of cells or organoids will expand
at a rate of about 4-5 fold per week or more than two population
doublings a week. Therefore, in some embodiments, the population of
cells or organoids will expand at a rate of at least 3 fold, at
least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at
least 8 fold, at least 9 fold or at least 10 fold per week.
[0314] Organoids of the invention may be obtained using cells
isolated from any suitable source. Generally, the cells used to
generate an organoid will be isolated from the same tissue type as
the organoid which is generated. The organoids are preferably
mammalian, for example, murine, bovine, porcine or human. Most
preferably, the organoids are human.
[0315] In some embodiments, the invention provides an organoid or
population of cells, wherein the organoid or population of cells is
a normal (healthy) organoid or population of cells or a disease
organoid or population of cells, for example obtained by culturing
stem cells taken from a human or animal with a disease.
[0316] In some embodiments, the invention provides an organoid or
population of cells which is frozen and stored at below -5.degree.
C., below -10.degree. C., below -20.degree. C., below -40.degree.
C., below -60.degree. C., or below -80.degree. C., below
-100.degree. C., below -150.degree. C. or at approximately
-180.degree. C. The organoid or population of cells of the
invention may be stored in liquid nitrogen. Therefore, in some
embodiments, the invention provides an organoid or population of
cells which is stored in liquid nitrogen.
[0317] In some embodiments, the invention provides an organoid of
the invention, wherein the organoid is a small intestine organoid,
a colon organoid, a gastric organoid, a pancreatic organoid, a
liver organoid or a prostatic organoid.
Organoid Structure and Morphology
[0318] Organoids of the invention, obtainable by expansion of stem
cells, provide a population of cells which resemble their in vivo
counterparts.
[0319] Image analysis may be used to assess characteristics of
cells in culture such as cell morphology; cell structures; evidence
for apoptosis or cell lysis; and organoid composition and
structure. Many types of imaging analysis are well known in the
art, such as electron microscopy, confocal microscopy,
stereomicroscopy, fluorescence microscopy. Histological analysis
can reveal basic architecture and cell types.
[0320] Illustrative examples of organoids generated according to
the invention are given in the accompanying figures. It can be seen
that organoids according to the invention may possess a layer of
cells with at least one bud and a central lumen. The organoids in
the outside of the matrigel tend to be larger than the organoids in
the center of the matrigel, perhaps because they have better access
to the necessary growth factors. Structurally, organoids according
to the invention are often elongated in shape. They may include one
or more budding structure--a single cell epithelial layer with
similarities to ducts or islets. Under confocal microscopy, the
structures may stain positive for keratin. They may include cells
with polarised nuclei and small cytoplasm. The organoids may have a
section which is formed of multiple layers; such cells often tend
to have their nuclei more central to the cells, i.e. not polarized.
The cells in the multilayer section may organise themselves to
include a gap, or lumen between the cells. In some embodiments the
organoids of the invention comprise or consist of epithelial cells.
In some embodiments, the organoids comprise or consist of a single
layer of epithelial cells. In some embodiments non-epithelial cells
are absent from the organoids. In some embodiments, the organoids
of the invention comprise all the differentiated cell types that
exist in their corresponding in vivo tissue counterpart.
[0321] In some embodiments human intestinal organoids displayed
budding organoid structures, rather than the cystic structures seen
under previous culture conditions. Metaphase spreads of organoids
more than 3 months old consistently revealed 46 chromosomes in each
of the 20 cells taken from three different donors.
[0322] In some embodiments the organoids of the invention comprise
a single monolayer of cells. In some embodiments the organoids of
the invention have a section which is formed of multiple layers.
Multiple layers of cells are also referred to herein as regions of
"stratified" cells. By "stratified" it is meant that there are
multiple (more than one) layers of cells. In some embodiments the
organoids of the invention comprise single monolayers that are
folded (or invaginated) to form two or more layers. It can
sometimes be difficult to distinguish between folded (or
invaginated) monolayers and regions of stratified cells. In some
embodiments an organoid comprises both regions of stratified cells
and regions of folded monolayers. In some embodiments the organoids
of the invention have a section which is formed of multiple layers
and a section comprising a single monolayer of cells.
Morphologically, the cells appear like their corresponding in vivo
tissue counterpart.
[0323] Therefore, in some embodiments the invention provides an
organoid, preferably obtainable using the culture media and methods
of the invention, which is a three-dimensional organoid comprising
epithelial cells surrounding a central lumen, wherein optionally
the epithelial cells exist in distinct dividing domains and
differentiating domains. In some embodiments the organoid of the
invention is a three-dimensional organoid comprising epithelial
cells arranged in regions of monolayers, optionally folded
monolayers and regions of stratified cells. In some embodiments,
non-epithelial cells are absent from said organoid. In some
embodiments, all differentiated cell types of the normal in vivo
tissue are present in said organoid.
Crypt-Villus Organoids
[0324] In small intestinal crypt-villus organoids the structural
arrangement of the organoids is very similar to the structure of in
vivo crypt-villi: the Lgr5+ stem cell and their niche cells (Paneth
cells) are next to each other at the base of the crypt, followed by
the transit amplifying cells, just above the base of the crypt and
leading into the sides of the villi and finally the differentiated
cells, such as enterocytes that make up the rest of the villi and
become more and more differentiated towards the top of the villi.
It can be seen that organoids according to the invention may
possess a layer of cells with at least one bud and a central lumen.
The organoids in the outside of the matrigel tend to be larger than
the organoids in the center of the matrigel, perhaps because they
have better access to the necessary growth factors. Structurally,
organoids according to the invention are often elongated in shape.
Under confocal microscopy, the structures may stain positive for
keratin. They may include cells with polarised nuclei and small
cytoplasm. The crypt-villus organoids are generally
single-layered.
[0325] In some embodiments, for example for a mouse crypt-villus
organoid, a crypt-villus organoid is a three-dimensional organoid,
comprising crypt-like domains surrounding a central lumen lined by
villus-like epithelial domains, which are epithelial domains
comprising differentiated cell types. In some embodiments,
non-epithelial cells are absent from said organoid.
[0326] In some embodiments, for example for a human crypt-villus
organoid, a crypt-villus organoid is a three-dimensional organoid,
comprising crypt-like domains surrounding a central lumen. In some
embodiments, dividing cells are confined to the budding structures.
No or few differentiated cells are present. Under differentiation
conditions the differentiated cells of the intestine are formed. In
some embodiments, non-epithelial cells are absent from said
organoid. In some embodiments, when the organoid is expanding, for
example when it is in an expansion culture medium according to the
invention, the organoid has few or no differentiated cells.
[0327] In some embodiments, a small intestinal organoid of the
invention cultured in a culture medium of the invention comprising
RANKL, comprises M-cells. In some embodiments of the invention, a
small intestinal organoid of the invention cultured in a culture
medium of the invention comprising a gamma-secretase inhibitor,
comprises goblet cells. In some embodiments, a small intestinal
organoid cultured in a differentiation medium (for example wherein
the differentiation medium comprises a basal medium, Noggin, EGF, a
TGF-beta inhibitor and a p38 inhibitor, a gamma-secretase inhibitor
and a RANKL) comprises all differentiated cell types including, for
example, goblet cells, neuroendocrine cells, tuft cells, M-cells,
enterocytes and paneth cells. Some of these differentiated cell
types, for example paneth cells, are also present (sometimes in
smaller quantities) in the expanding organoids.
[0328] Human intestinal organoids display budding organoid
structures, rather than the cystic structures seen under previous
culture conditions. The upper opening of freshly isolated crypts
becomes sealed and this region gradually balloons out and becomes
filled with apoptotic cells, much like apoptotic cells are pinched
off at the villus tip. Thus, in some embodiments, the crypt-villus
organoids have a crypt-like structure surrounding a central lumen
lined by a villus-like epithelium and filled with apoptotic cell
bodies. In some embodiments, the lumen is opened at consecutive
time intervals to release the content into the medium.
[0329] In some embodiments, the crypt region undergoes continuous
budding events which create additional crypts, a process
reminiscent of crypt fission.
[0330] The inventors also demonstrated that the human intestinal
organoids generated by media and methods of the present invention,
mimicked in vivo cell fate decisions in response to external
factors. For example, it has previously been shown that Notch
inhibition in intestinal stem cells, terminates intestinal
epithelial proliferation and induces goblet cell hyperplasia in
vivo. Thus in some embodiments, when a crypt-villus organoid of the
invention is treated with a Notch inhibitor, proliferation ceases
and most cells (for example more than 50%, more than 60%, more than
70%, more than 80%, more than 90%, more than 95%, more than 98%)
convert into goblet cells within 3 days.
[0331] Metaphase spreads of organoids more than 3 months old
consistently revealed 46 chromosomes in each of the 20 cells taken
from three different donors. Furthermore, microarray analysis
revealed that the stem cells in culture possessed similar molecular
signatures to intestinal crypt cells including intestinal stem cell
genes.
Colon Organoids
[0332] Colon organoids exhibit a similar cell composition to
crypt-villus organoids. Thus, the comments for crypt-villus
organoids above apply to colon organoids mutatis mutandis. For
example, see FIGS. 1 and 2.
[0333] Typically, the difference between the colon and small
intestinal organoids is that the crypts are shallower in the colon
making it look a little like a "football" rather than a sphere with
protrusions. Both small intestinal and colon organoids have domains
that contain stem cells and transit amplifying (TA) cells, and
other domains containing differentiating and/or differentiated
cells. For the small intestinal organoids the differentiated
domains are sometimes referred to as "villus-like". The
differentiated domains of the colon organoids are typically similar
in cell composition to the "villus-like" domains of the small
intestine but the colon itself does not have villi.
[0334] The amount of Wnt present can influence the size of the
budding structures (i.e. the depth of the crypts) in the organoids.
More Wnt reduces budding. The colon produces more Wnt than the
small intestine and so requires less additional Wnt in the culture
medium and typically has shallower crypts than the small intestine.
The same difference is seen in the organoids.
[0335] In some aspects, colon organoids are provided by the
invention. The inventors have found that mouse colon organoids can
be obtained by culturing colon crypts in an ENR+Wnt3A (WENR) cell
culture media. Thus, in some embodiments, the invention provides a
colon organoid obtained by culturing colon crypts in WENR
media.
[0336] The inventors have also surprisingly found that human colon
organoids can be maintained using a culture medium comprising WENR
plus gastrin plus nicotinamide. In some embodiments, a human colon
organoid of the invention is obtainable by using a media comprising
WENR plus gastrin plus nicotinamide and also comprising an
inhibitor of TGF beta. For example, in some embodiments, the
following cell culture media may be used to obtain a human colon
organoid: WENR+ gastrin+ nicotinamide+A8301+SB202190. In other
embodiments, the following cell culture medium may be used to
obtain a human colon organoid: WENR+Nicotinamide+A83-01
[0337] In some embodiments, a mouse colon organoid has a maximal
diameter of approximately 200-700 um, for example 250-600 um,
300-500 um, 320-450 um, 340-400 um, 300-380 um, for example
approximately 360 um. In some embodiments, a colon organoid has a
minimal diameter of approximately 100-400 um, for example 150-350
um, 170-300 um, 190-280 um, 195-250 um, for example, approximately
235 um. In a further embodiment, the organoids can have a diameter
of up to 1 mm. In some embodiments, a human colon organoid has a
maximal diameter of approximately 300-800 um, for example 350-700
um, 400-600 um, 450-550 um, 475-540 um, 500-530 um, for example
approximately 500 um. In some embodiments, a colon organoid has a
minimal diameter of approximately 200-500 um, for example 250-450
um, 300-415 um, 350-400 um, 325-380 um, for example, approximately
375 um. In a further embodiment, the organoids can have a diameter
of up to 1 mm. In some embodiments, a colon organoid of the
invention comprises budding structures. These may be visible by
using EdU stain to visualize proliferating cells.
[0338] Human colon organoids retain their characteristic budding
structure under the Human Intestinal Stem Cell Culture ("HISC")
condition (WENRg+ nicotinamide+TGF-beta inhibitor (e.g. A83-01)+p38
inhibitor (e.g. SB202190)), In some embodiments, a colon organoid
is a three-dimensional organoid, comprising budding structures
which are proliferating and contain stem cells. These stem cell
domains surround a central lumen. Dividing cells are generally
confined to the budding structures. In some embodiments, no or few
differentiated cells are present. Under differentiation conditions
the differentiated cells of the intestine are formed, for example
mature enterocytes. In some embodiments, non-epithelial cells are
absent from said organoid.
Pancreatic Organoids
[0339] Pancreatic organoids of the invention preferably exhibit
budding. In some embodiments, the pancreatic organoids are from
100-1000 micrometers in diameter, for example, 200-900 micrometers,
300-1000 micrometers, 400-700 micrometers. The pancreatic organoids
are preferably single layered. There are only the very beginnings
of islet or ductal structures. Budding structure are indicative of
a healthy proliferation status and stem cell maintenance.
[0340] In some embodiments, for example when pancreatic organoids
are grown in a culture medium of the invention (and in the absence
of TGF-beta inhibitors), pancreatic organoids are mainly cystic
structures with few budding structures or duct-like domains. The
cystic structures comprise mainly monolayers but some regions of
stratified cells may be present. The cells express stem cell and
progenitor (ductal) markers. No differentiated cells, such as
beta-cells, are present in the organoids. The cyst is mainly formed
by a monolayer, but stratified parts exist. Cell types resemble
stem cells/progenitor (duct cell gene expression). There are no
differentiated cells ((3-cells).
[0341] In other embodiments, for example when pancreatic organoids
are grown in the presence of a TGF-beta inhibitor, such as A83-01,
for example in a culture medium of the invention, the pancreatic
organoids comprise more budding structures/ductal-like domains
(this means cells are duct-like cells more than the structure is
like a duct), as shown by Krt 19 staining (for example, see FIG.
31). Monolayers of polarized cells can be identified, but also
areas with stratified cells.
Adenocarcinoma and Colon Cancer Organoids
[0342] Adenocarcinoma and colon cancer organoids generally form
cystic structures instead of budding structures. This is
reminiscent of the absence of good cell niche support. Adenoma
crypts cultured with EFG+Noggin show approximately
16.times.expansion in the first 10 days. Adeno(carcino)ma and colon
cancer organoids may provide useful research tools and drug
screening models.
[0343] Carcinoma, adenoma and adenocarcinoma organoids are largely
cystic (for example, see FIGS. 4 and 9). However, in some
embodiments, they may also comprise structures that resemble their
normal tissue organoid counterparts.
Barrett's Esophagus (BE) Organoids
[0344] A BE organoid of the invention comprises budding structures
(for example, see FIG. 5).
[0345] Morphologically, the cells in the organoids of the invention
appear like their corresponding in vivo tissue counterpart.
[0346] Barrett's Esophagus is a disease marked by the presence of
columnar epithelium in the lower esophagus, replacing the normal
squamous cell epithelium as a result of metaplasia. The
histological hallmark of Barrett's esophagus is the presence of
intestinal goblet cells in the esophagus. Exploiting the similarity
between Barrett's Esophagus and the intestinal epithelium, the
inventors showed that the culture medium and methods of the
invention could be used to maintain Barrett's Esophagus epithelium
for up to 1 month. The inventors also demonstrated, for the first
time, that addition of FGF10 to the culture medium of the invention
enabled the Barrett's Esophagus organoids to form budding
structures and significantly prolonged the culture duration to more
than three months. Thus, a Barrett's Esophagus organoid is an
example of an organoid of the invention. In some embodiments, a
Barrett's Esophagus organoid has a cystic structure. In some
embodiments, a Barrett's Esophagus organoid of the invention
comprises Paneth cells. In some embodiments, a Barrett's Esophagus
organoid of the invention expresses lysozyme.
[0347] The inventors, therefore, also describe a culture medium
according to the invention, comprising FGF10, for the culture of
Barrett's Esophagus epithelium.
[0348] In some embodiments of the invention, Barrett's Esophagus
organoids may be grown using a culture medium according to
invention also comprising FGF10. In some embodiments, these
Barrett's Esophagus organoids express Ki67 and have a minimal
number, preferably less than 10%, less than 5% or less than 1%
PAS-positive cells and Mucin-positive cells. In some embodiments,
the Barrett's Esophagus organoids comprise lysozyme-positive Paneth
cells.
Stomach (Gastric) Organoids (for Example, See FIG. 46)
[0349] Mouse gastric organoids grown in a culture medium of the
invention are three-dimensional organoids, comprising or consisting
of a single layer epithelia, that comprises a gastric gland base
like domains (formed by stem and progenitor cells) surrounding a
central lumen lined by epithelial domains comprising differentiated
cell types, and optionally wherein non-epithelial cells are absent
from said organoid.
[0350] Human gastric organoids grown in a culture medium of the
invention comprise cystic structures. The cystic structure is a
monolayer of polarized cells. These human gastric organoids grown
in the presence of a TGF-beta inhibitor resemble mouse gastric
organoids much more closely than human organoids grown in the
absence of TGF-beta inhibitor.
Prostatic Organoids (See FIGS. 41 to 43)
[0351] Under culture conditions comprising EGF, Noggin, Rspondin,
murine prostatic organoids form three dimensional cystic structures
with a lumen. In time the layers fold inward forming 3-4 layers of
(stratified) epithelial cells. The outer layer is mostly composed
of CK5+ basal epithelial cells whereas the inner layers are mostly
composed of CK8+ luminal epithelial cells. No stem cell compartment
has been identified; all domains contain dividing cells. Therefore,
in some embodiments, a prostate organoid grown in the absence of
testosterone comprises stratified layers of dividing epithelial
cells. In a further embodiment, the prostate organoid comprises an
outer layer of cells comprising CK5+ basal epithelial cells and
inner layers comprising CK8+ luminal epithelial cells. In some
embodiments, a prostate organoid grown in the absence of
testosterone does not contain any stem cells.
Addition of Testosterone to the Prostate Culture Medium
[0352] The inventors have shown that the addition of (DiHydro)
testosterone to the culture conditions for the prostatic organoids,
results in the majority of cells differentiating into CK8+ luminal
cells which form a single layer of epithelium that folds onto
itself into two layers. Prostate organoids grown in the presence of
testosterone consist of mostly luminal cells with or without a
second layer of basal cells. The structure resembles the in vivo
structure. Both differentiated and dividing cells are present, as
well as stem cells and progenitors. Therefore, in some embodiments,
for example when cultured in a medium comprising testosterone, a
prostate organoid is a three-dimensional organoid comprising cystic
structures and a lumen. In some embodiments the prostate organoid
comprises CK8+ luminal cells which form a monolayer of epithelium.
In some embodiments the monolayer is folded into two or more
layers. In other embodiments, the organoid may comprises regions of
stratified cells. In some embodiments, the prostate organoid
comprises differentiated cells while maintaining the dividing stem
cell population. In some embodiments, the shape of the organoid is
determined by the origin of the cellular or tissue starting
material (i.e. the position in the prostate before isolation). The
prostate consists of different lobes or regions which display the
different epithelial structures described above (stratified and
folded), After in vitro culturing the organoids appear to some
extent to maintain the different macroscopic structure (stratified
or folded) of the part of the prostate from which it
originated.
Liver Organoids
[0353] Structurally, mouse liver organoids according to the
invention are often elongated in shape. They may include one or
more budding structure--a single cell epithelial layer which has a
structure not unlike a bile duct. Under confocal microscopy, the
structures may stain positive for keratin. They may include cells
with polarised nuclei and small cytoplasm. The organoids may have a
section which is formed of multiple layers; such cells often tend
to have their nuclei more central to the cells, i.e. not polarized.
The cells in the multilayer section may organise themselves to
include a gap, or lumen between the cells. Human liver organoids of
the invention, in some embodiments have a generally cystic
structure.
[0354] In some embodiments, a liver organoid is a three-dimensional
organoid, with a cystic structure (for example, see FIG. 30). Under
expansion conditions the organoid may consist of stem cells and
progenitor cells where two domains are defined: (1) A duct-like
domain, formed by a single-layer cubical epithelia (positive for
the ductal marker Krt19) with cells lining a central lumen; and (2)
a pseudo-stratified epithelial domain where krt19 positive cells
and scattered albumin positive cells are detected. This
architecture (areas with single layer epithelia together with areas
of pseudostratified epithelia) resembles the embryonic liver bud.
Under expansion conditions fully differentiated cells are not
present, although expression of hepatocyte/hepatoblast-specific
markers can in some embodiments be detected. Differentiation
conditions result in the formation of a cystic organoid where the
duct-like domain (single layer epithelia) is lost and the entire
structure becomes a pseudo-stratified epithelia containing >50%
polarized hepatocytes.
[0355] A liver organoid, preferably comprises a hepatocyte and a
cholangiocyte cell (although hepatocytes are especially seen
following differentiation in DM and are not required for
expansion), more preferably wherein at least one of the following
markers could be detected: at least one hepatocyte marker such as
albumin, transthyretrin, B-1 integrin and Glutamine synthetase
and/or at least one of CYP3A11, FAH, tbx3, TAT and Gck and/or at
least one cholangiocyte maker such as Keratin 7 and 19. The skilled
person knows how to detect each of these markers (i.e. RT-PCR
and/or immunofluorescence). Preferably the expression of each of
these markers is assessed as carried out in the experimental part.
Each of these markers is usually expressed after at least two
weeks, three weeks or one month of culture using a method of the
invention. Microarray analysis of the organoids in both culture
conditions showed that liver organoids resemble adult liver
tissue.
[0356] Preferably all cells in a liver organoid express hepatocyte
surface markers. For example, in some embodiments, at least 50%
(for example 50-60%), at least 60%, at least 70%, at least 80%, at
least 90%, at least 99% or 100% of cells in a liver organoid
express hepatocyte markers. In some embodiments, approximately 35%
of the cells in a liver organoid express a hepatocyte surface
marker, for example, 25-45%, 30-40%, 33-37%, 35% or less, or 15-35%
of cells. In some embodiments, the expansion phase would have less
hepatocytes, for example less than 20%, less than 10%, less than 5%
of the cells, less than 2%, less than 1%, preferably 0% of the
cells. Preferably, cells and organoids generated according to the
invention also possess hepatocyte functions, such as expressing or
staining positive for the mature hepatic markers albumin, B-1
integring, CK-8, CK-18, transthyretin (TTR), glucose 6P, Met,
Glutamine synthase (Glul), transferrin, Fand1, Fand2a, K7, K19 and
cytochrome P450 isoforms 3A13 (CYP3A13), 51 (CYP51) 2D10 (CYP2D10),
2j6 (CYP2j6), 39A1 (CYP39A1), 4A10 (CYP4A10), 4F13 (CYP4F13) 4F16
(CYP4F16), CYP4B1 and 20A1 (CYP20A1). Also, embryonic liver gene
AFP is in some embodiments not detected in neither of both culture
conditions, as in adult liver. In some embodiments, the expression
of alpha fetal protein is just above the background gene
expression.
[0357] Also, the well-known liver transcription factors as HNF1a,
HNF1b and HNF4a are highly expressed in both conditions.
[0358] Since liver and pancreas are closely related organs, we
investigated whether our liver cultures also expressed
pancreas-specific genes. The pancreas is functionally divided into
endocrine and exocrine pancreas. The endocrine pancreas is mainly
characterized for expressing insulin, glucagon and somatostatin.
The expression of these hormones is tightly regulated by a set of
endocrine pancreas-specific transcription factors, the most
important being Pdx1 and NeuroD. The exocrine pancreas is formed by
acinar and ductal compartments responsible of producing the
digestive enzymes amylase, pancreatic lipase and chymotrypsin,
among others. The expression of these genes is also regulated by
specific exocrine pancreatic genes as Ptf1.
[0359] The pancreas specific genes Ptf1a, pancreatic amylase
(Amy2a4), pancreatic lipase (Pnlip), insulin (ins1 and ins2),
glucagon (Gcg), chymotrypsin (cela1), Pdx1 and NeuroD were absent
in the liver cultures here described.
[0360] In some embodiments, one or more or all of the following
genes are expressed in the liver organoids at a similar level to
the corresponding gene in adult liver hepatocytes: Aqp1, Bmp1,
Apo3, Apol7a, Sord, C3, Ppara, Pparg, tbx3, lgf1, ll17rb, l11b,
Tgfbi, Apoa1, Apoa4, Apob, Cyp26b1, Cyp27a1, Cyp2b13, Cyp2b9,
Cyp2c37, Cyp2f2, Cyp2g1, Cyp2j13, Cyp3a11, Cyp4a10 and Cypf14. For
example, see FIG. 27A.
[0361] In some embodiments, one or more of the following genes is
expressed in the liver organoids at a similarly shut down level
compared to the corresponding gene in adult liver hepatocytes:
Cc12, Osmr, Icam1 and Cxcl2.
[0362] In some embodiments, one or both of the following genes is
differentially expressed in both a liver organoid and newborn
liver: mKi67 and cdkn3, meaning that the expression of these genes
is higher in the organoids than in the differentiated organoids or
whole organ.
[0363] In some embodiments, one, two or all of the following genes
are expressed at a similar level in a liver organoid and a newborn
liver: cyp2j6, olfm4 and Lefty 1. For example, see FIG. 27B.
[0364] In some embodiments, a liver organoid of the invention has a
ductal phenotype when cultured in expansion medium of the invention
(e.g. EM1 or EM2).
[0365] In some embodiments, a liver organoid of the invention
expresses adult liver markers when cultured in a differentiation
medium of the invention.
[0366] In one embodiment, a liver organoid of the invention has a
gene expression profile as shown in FIG. 27C.
[0367] In a particularly preferred embodiment, a mouse liver cell
population or organoid of the invention has the gene expression
profile as shown in FIG. 28. For example, in one preferred
embodiment, a mouse liver cell population or organoid of the
invention: [0368] a) expresses at least one (e.g. 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11), preferably all of the following stem cell
markers: lgr5, lgr4, epcam, Cd44, Tnfrsf19, Sox9, Sp5, Cd24a,
Prom1, Cdca7 and Elf3; and/or [0369] b) does not express the
following stem cell marker: lgr6; and/or [0370] c) expresses at
least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19), preferably all of the following hepatocyte or
cholangiocyte markers when grown in expansion medium of the
invention: Hnf1a, Hnf1b, Hnf4a, Hhex, Onecut1, Onecut2, Prox1,
Cdh1, Foxa2, Gata6, Foxm1, Cebpa, Cebpb, Cebpd, Cebpg, Glu1, Krt7,
Krt19 and Met; and/or [0371] d) does not express at least one (e.g.
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) of the
following genes when grown in expansion medium of the invention:
afp, Ins1, Ins2, Gcg, Ptf1a, Cela1, Cela2a, Cela3b, Neurod1,
Neurod2, Neurog1, Neurog2, Neurog3, Amy2a4, Igf1r, Igf2 and Cd34;
and/or [0372] e) expresses at least one (e.g. 1, 2 or 3) of the
following reprogramming genes: Klf4, Myc and Pou5f1 and/or [0373]
f) does not express the following reprogramming gene: Sox2. [0374]
wherein the expression of the genes is preferably detected by
measuring expression at the mRNA level, for example, using a
microarray.
[0375] More preferably a mouse liver cell population or organoid of
the invention has all of features a) to f) above.
[0376] In some embodiments, the gene expression profile described
above for a mouse liver cell population or liver organoid of the
invention is for a mouse cell population or organoid cultured in
liver expansion medium of the invention.
[0377] In some embodiments, there is provided a human liver cell
population or organoid of the invention that has the gene
expression signature shown in FIG. 29. For example, a human liver
cell population or organoid cultured in EM1 of the invention
preferably expresses the genes indicated in FIG. 29 as being
expressed in EM1 cell culture medium. For example, a human liver
cell population or organoid cultured in EM2 of the invention
preferably expresses the genes indicated in FIG. 29 as being
expressed in EM2 cell culture medium. For example, a human liver
cell population or organoid cultured in DM of the invention
preferably expresses the genes indicated in FIG. 29 as being
expressed in DM cell culture medium.
[0378] For example, in one preferred embodiment, a human liver cell
population or organoid of the invention: [0379] a) expresses at
least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9), preferably all of the
following stem cell signature genes: LGR4, TACSTD1/Epcam, CD44,
SOX9, SP5, CD24, PROM1, CDCA7 and ELF3; and/or [0380] b) expresses
at least one (e.g. 1, 2, 3, 4), preferably all of the following
reprogramming genes: KLF4, MYC, POU5F1 and SOX2; and/or [0381] c)
expresses at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19), preferably all of the following
hepatocyte/cholangiocyte specific genes: HNF1A, HNF1B, HNF4A, HHEX,
ONECUT1, ONECUT2, PROX1, CDH1, FOXA2, GATA6, FOXM1, CEBPA, CEBPB,
CEBPD, CEBPG, GLUL, KRT7, KRT19 and MET; and/or [0382] d) does not
express at least one (e.g. 1, 2, 3, 4, 5, 6), preferably all of the
following hepatocyte/cholangiocyte specific genes: NEUROG2, IGF1R
and CD34, AFP, GCG and PTF1A, for example, it does not express
NEUROG2, IGF1R and CD34; and/or [0383] e) expresses at least one
(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18), preferably all of the following hepatocyte specific genes:
TTR, ALB, FAH, TAT, CYP3A7, APOA1, HMGCS1, PPARG, CYP2B6, CYP2C18,
CYP2C9, CYP2J2, CYP3A4, CYP3A5, CYP3A7, CYP4F8, CYP4V2 and
SCARB1;
[0384] wherein the expression of the genes is preferably detected
by measuring expression at the mRNA level, for example, using a
microarray.
[0385] More preferably a human liver cell population or organoid of
the invention has all of features a) to e) above.
[0386] In some embodiments, the genes in a human liver cell
population or organoid of the invention are upregulated or
downregulated relative to expression of a reference RNA as shown in
FIG. 29. Preferably, the reference RNA is Universal Human Reference
RNA (Stratagene, Catalog #740000). In some embodiments, a gene is
upregulated or downregulated relative to the reference RNA if it is
also shown in FIG. 29 as being upregulated or downregulated
relative to the reference RNA but the extent of upregulation or
downregulation need not be the same. In other embodiments, the
extent of upregulation or downregulation is +/-35%, +/-30%, +/-25%,
+/-20%, +/-20%, +/-15%, +/-10%, +/-5%, +/-3%, or more preferably
+/-1.5-fold, +/-2-fold, +/-3-fold, +/-5-fold or approximately the
same as shown in FIG. 29. In other embodiments, the absolute level
of expression of the genes in a human organoid of the invention is
+/-35%, +/-30%, +/-25%, +/-20%, +/-15%, +/-10%, +/-5%, +/-3%, or
+/-1.5-fold, +/-2-fold, +/-3-fold, +/-5-fold or approximately the
same as shown in FIG. 29.
[0387] The human liver cell population or organoids of the
invention also preferably express Lgr5 and/or Tnfrsf19, preferably
both. In some embodiments, the human liver cell population or
organoids, when cultured in expansion medium of the invention
express Lgr5 and/or Tnfrsf19, preferably both. Preferably,
expression of Lgr5 and/or Tnfrsfr19 is detected by RT PCR. In some
embodiments, Lgr5 and/or Tnfrsf19 are present at much lower levels
of expression in organoids or cells when cultured in the
differentiation medium compared to their level of expression
organoids or cells when cultured in the expansion medium (for
example at least 2-fold, at least 3-fold, at least 4-fold, at least
5-fold, at least 10-fold, at least 15-fold lower).
[0388] Liver cells and organoids according to the present invention
may preferably be capable of secreting albumin, for example, at a
rate of between approximately 1 .mu.g per hour per 10.sup.6 cells
and 10 .mu.g per hour per 10.sup.6 cells, preferably between 2
.mu.g and 6 .mu.g per hour per 10.sup.6 cells.
[0389] Furthermore, such liver cells and organoids may secrete
urea. For example, in a 35 mm dish of cells, the activity of urea
synthesis may be between 1 .mu.g and 50 .mu.g in 48 hours,
preferably between 5 .mu.g and 30 .mu.g.
[0390] Liver cells and organoids according to the invention may
show visible glycogen stores, for example, when stained. The
capacity for cells and organoids according to the invention to
synthesize glycogen actively can be tested by switching the culture
media from low-glucose differentiation media to high-glucose DMEM
supplemented with 10% FBS and 0.2 .mu.M dexamethasone for two
days.
[0391] Liver cells and organoids according to the invention may
possess inducible cytochrome P450 activity (e.g. CYP1A). Such
activity may be tested, for example, using an
ethoxyresorufin-O-deethylase (EROD) assay (Cancer Res, 2001, 61:
8164-8170). For example, cells or organoids may be exposed to a
P450 substrate such as 3-methylcholanthrene and the levels of EROD
activity compared to control cells.
[0392] Morphologically, the liver organoid cells appear
hepatocyte-like.
[0393] A preferred liver organoid comprises or consists of a cystic
structure with on the outside a layer of cells with buds and a
central lumen as depicted in FIG. 30. This liver organoid may have
one or more (e.g. 2, 3, or all 4) of the following characteristics:
(a) having a cell density of >5.times.10.sup.5 cells/cm.sup.3,
preferably >10.times.10.sup.5 cells/cm.sup.3; (b) having a
thickness equivalent to 2-30 layers of cells, preferably a
thickness equivalent to 2-15 layers of cells; (c) the cells
mutually contact in three dimensions, (d) demonstrate a function
inherent to healthy liver tissue, (e) have an elongated shape, with
2 defined domains, i.e. a single layered epithelial domain where
highly polarized cells are detected and keratin markers are
expressed (this domain resembles the bile duct domain) and the
other domain constitutes the main body of the organoid and is
formed by a multilayered epithelia with non-polarized cells wherein
albumin expression may be detected. It is clear to the skilled
person that such a liver organoid is preferably not a liver
fragment and/or does not comprise a blood vessel, and/or does not
comprise a liver lobule or a bile duct.
[0394] Within the context of the invention, a liver fragment is a
part of an adult liver, preferably a human adult liver. Preferably
a liver organoid as identified herein is therefore not a liver
fragment. A liver organoid is preferably obtained using a cell from
an adult liver, preferably an epithelial stem cell from an adult
liver, more preferably an epithelial stem cell from an adult liver
expressing Lgr5. A liver organoid may also be obtained from any
cell which upon damage or culturing expresses Lgr5 and is therefore
an Lgr5-expressing cycling stem cell.
[0395] In some embodiments, a liver organoid comprises cells that
express Lgr5. For example, in some embodiments, at least 2%, more
preferably at least 5%, at least 10%, at least 20%, at least 30%,
at least 40%, at least 50%, at least 60%, at least 70%, at least
80%, at least 90%, at least 95% of the cells in the liver organoid
express Lgr5. Similarly, the invention provides a cell or a
population of cells which express Lgr5, wherein said cells are
obtained from a liver organoid of the invention. The progeny of
such cells is also encompassed by the invention.
[0396] In an embodiment, a liver organoid is a liver organoid which
is still being cultured using a method of the invention and is
therefore in contact with an extracellular matrix. Preferably, a
liver organoid is embedded in a non-mesenchymal or mesenchymal
extracellular matrix. Within the context of the invention, "in
contact" means a physical or mechanical or chemical contact, which
means that for separating said liver organoid from said
extracellular matrix a force needs to be used.
[0397] In a preferred embodiment, a liver organoid could be
cultured during at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 months or longer. In some embodiments, the
liver organoid is expanded or maintained in culture for at least 3
months, preferably at least 4 months, at least 5 months, at least 6
months, at least 7 months, at least 9 months, or at least 12 months
or more. Preferably, a liver organoid cultured using expansion
media of the invention comprising a TGF beta inhibitor may be
cultured for at least 4 weeks, more preferably at least 5 weeks at
5 fold expansion a week or two or more population doublings per
week (e.g. for at least 10 doublings, at least 20 doublings, more
preferably at least 25 doublings, for example, at least 30
doublings). Preferably, a liver organoid cultured using expansion
media of the invention comprising a prostaglandin pathway activator
in addition to a TGF beta inhibitor may be cultured for at least 7
weeks, more preferably at least 8 weeks at 2 or more doublings
(e.g. 2-3 doublings) per week (i.e. at least 15 doublings, at least
25 doublings, at least 30 doublings, at least 32 doublings, at
least 35 doublings, e.g. 32-40 doublings or at least 40 doublings,
for example, at least 50 doublings). Thus, preferably, a liver
organoid of the invention, for example a human liver organoid, is
obtained using expansion media of the invention.
[0398] In another preferred embodiment, a liver organoid originates
from a single cell, preferably expressing Lgr5, more preferably
wherein the single cell comprises a nucleic acid construct
comprising a nucleic acid molecule of interest.
Organoid Composition and Gene Expression
[0399] The crypt-villus, colon crypt and pancreatic organoids
typically comprise stem cells and/or progenitor cells and,
therefore, these organoids share certain patterns of gene
expression. In some embodiments, one or more (for example, 1, 2, 3,
4, 5, 6 or 7) or all of the following markers can be detected:
LGR5, LGR4, epcam, Cd44, Sox9, Cd24a, and CD133/Prom1 and
optionally Tnfrsf19. In another embodiment, the expression of one
or two or all of the following progenitor genes can be detected:
Pdx1, Nkx2.2, and Nkx6.1. After differentiation, gene expression
patterns of the crypt-villus, colon crypt and pancreatic organoids
are expected to diverge as the differentiated organoids express
tissue-specific adult markers, such as insulin in the pancreas for
example.
Crypt Villus Organoids
[0400] In some embodiments of the invention, the organoids comprise
crypt-villus like extensions which comprise all differentiated
epithelial cell types, including proliferative cells, Paneth cells,
enterocytes and goblet cells. In some embodiments, the crypt-villus
organoids of the invention do not contain myofibroblasts or other
non-epithelial cells. A crypt-villus organoid of the invention
preferably comprises enterocytes, including absorptive enterocytes,
goblet cells, enteroendocrine cells, and Paneth cells in a
crypt-villus-like structure. Preferably at least one (for example,
2, 3, 4, 5 or 6) of the following markers could be detected: SMOC2,
CDCA7, OLFM4, ASCL2, AXIN2 and/or Lgr5 Tnfrsf19, CD24a, Sox9, CD44,
Prom1 (see FIG. 2e and FIG. 14). In some embodiments, the markers
RNF43 and ZNRF3 can be detected. In some embodiments, one or more
(for example 1, 2, 3, 4 or 5) or all of SMOC2, CDCA7, OLFM4, ASCL2,
AXIN2 and/or Lgr5 are at least 2-fold, 3-fold, or 4-fold
upregulated in crypts, whereas markers that are at least 2-fold,
3-fold, or 4-fold downregulated in crypts include at least one or
more (for example 1, 2, 3 or 4) or all of ABCG1, ENPP3, CSTE, MUC17
and/or APOA1. In this context "upregulation" is relative to the
villus of the intestine or to the top section of the colon crypt.
Microarray analysis, comparing the gene expression of
differentiated organoid cells to stem cells, revealed that the
small intestinal crypt-villus and colonic organoids possess
comparable molecular signatures of intestinal crypts including the
expression of intestinal stem cell genes. Thus, the invention also
provides a colonic organoid having the molecular signature
described above for crypt-villus organoids. Organoids cultured
in-vitro clearly exhibit a similar expression profile to freshly
isolated small intestinal crypts and express known stem cell
markers.
[0401] In some embodiments, the mRNA encoding one or more genes
(e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24 or 25) listed in FIG. 14 (for example
all of the genes shaded in FIG. 14) as being upregulated in
crypt-villus organoids or colon organoids respectively is
upregulated in a crypt-villus organoid or colon organoid of the
invention compared to a freshly isolated small intestinal villi, as
determined by microarray. In some embodiments, the mRNA encoding
one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25) genes listed in FIG.
14 (for example all of the genes shaded in FIG. 14) as being
downregulated in crypt-villus organoids or colon organoids
respectively is downregulated in a crypt-villus organoid or colon
organoid of the invention compared to a freshly isolated small
intestinal villi, as determined by microarray. In some embodiments,
the fold upregulation or downregulation is as indicated in FIG.
14+/-25%, for example, +/-20%, +/-15%, +/-10%, +/-5%, +/-3% or
approximately as quoted in FIG. 14. For example, a crypt-villus
organoid of the invention may have ADORA2B upregulated 9.54 fold
+/-25% compared to freshly isolated small investinal villi. The
same applies, mutatis mutandis, to the other genes listed in FIG.
14.
[0402] In some embodiments, the crypt villus organoids show natural
expression of Lgr5. In some embodiments, the crypt villus organoids
show natural expression of at least Lgr5 and one or more (for
example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) or
all of stem cell markers from the group consisting of: CK19,
Nestin, Somatostatin, CXCR4.sup.+, CD133.sup.+, DCAMKL-1, CD44,
Sord, Sox9, CD44, Prss23, Sp5, Hnf1.alpha., Hnf4a, Sox9, KRT7 and
KRT19. In addition or alternatively, crypt-villus organoids may be
characterised by expression of one or more or all (for example 1 or
2) of: Sord and/or Prss23. In addition or alternatively,
crypt-villus organoids may be characterised by expression of CD44
and/or Sox9. In another embodiment, the crypt-villus organoids show
expression of one or more (for example 1, 2, 3, 4, 5, 6, 7, 8, 9)
or all of the markers from the group consisting of: lgr5, lgr4,
epcam (tacstd1), Cd44, Tnfrsf19, Sox9, Sp5, Cd24a, Prom1, and
Cdca7.
[0403] In some embodiments, a crypt villus organoid comprises
Paneth cells expressing lysozyme.
Colon Organoids
[0404] In some embodiments, a colon organoid contains
enteroendocrine cells (e.g. as detectable using chromagranin A
stain), goblet cells (as detectable using mucin 2 stain). In some
embodiments, less than 10% of the cells in the colon organoid are
enteroendocrine cells (e.g. 0.01-5%, 0.1-3%).
[0405] In some embodiments, less than 30% of the cells in the colon
organoid are goblet cells (e.g. 1-25%, 1-15%, 5-10%). In some
embodiments, the distribution of the enteroendocrine cells and/or
the goblet cells is as shown in the FIG. 1d.
[0406] In some embodiments, a colon organoid contains mature
enterocytes (e.g. as visualised by alkaline phosphatise staining).
In some embodiments, less than 10% of the cells in the colon
organoid are mature enterocytes (e.g. less than 5%, less than 3%,
0.01-5%, 0.1-3%, 0.1-5%).
[0407] In preferred embodiments, a colon organoid does not comprise
Paneth cells because there are no Paneth cells in a naturally
occurring in vivo colon.
[0408] In some embodiments, the colon organoids show natural
expression of Lgr5.
[0409] In some embodiments, a colon organoid expresses one or more
(e.g. 1, 2, 3 or 4) of Villin1, Alpi, ChgA and Muc2. In some
embodiments, the relative amount of Villin1 mRNA expressed by a
colon organoid of the invention compared to a freshly isolated
colon crypt is at least 3% (e.g. at least 5%, at least 8%, at least
10%), for example between 5-15%. In some embodiments, the relative
amount of Alpi mRNA expressed by a colon organoid of the invention
compared to a freshly isolated colon crypt is at least 0.5% (e.g.
at least 1%, at least 2%), for example, between 0.5-5%. In some
embodiments, the relative amount of ChgA mRNA expressed by a colon
organoid of the invention compared to a freshly isolated colon
crypt is at least 15% (e.g. at least 20%, at least 22%), for
example, between 15-30%. In some embodiments, the relative amount
of Muc2 mRNA expressed by a colon organoid of the invention
compared to a freshly isolated colon crypt is at least 20% (e.g. at
least 25%, at least 30%, at least 35%), for example, between
25-37%.
[0410] In some embodiments, a human colon organoid of the invention
expresses known stem cell markers.
Pancreatic Organoids
[0411] The pancreas contains three classes of cell types: the
ductal cells, the acinar cells, and the endocrine cells. The
endocrine cells produce the hormones glucagon, insulin somatostatin
and pancreatic polypeptide (PP), which are secreted into the blood
stream and help the body regulate sugar metabolism. The acinar
cells are part of the exocrine system, which manufactures digestive
enzymes, and ductal cells from the pancreatic ducts, which connect
the acinar cells to digestive organs. During development, Islets of
Langerhans are thought to descend from progenitor endocrine cells
which emerge from the pancreatic duct and after differentiation
aggregate to form Islets of Langerhans. Islets of Langerhans
comprise .alpha. cells, .beta. cells, .delta. cells, and PP
cells.
[0412] Pancreatic organoid cells may have an expression pattern
that resembles ductal cell markers, such as one or more (e.g. 1, 2
or all) of K7, K19 and Hnf1b and/or one or more general stem cell
markers such as Sox9 and/or Onecut1. This is likely to be part of
their stem cell signature. Generally, fewer differentiation markers
are seen. In some embodiments in which a cell is isolated from a
pancreatic duct in order to generate a pancreatic organoid of the
invention, the cell type that gives rise to a pancreatic organoid
of the invention is not a ductal cell (meaning the epithelial cells
positive for keratin 7 and keratin 19 that form the ductal tube),
but it is a cell attached to the pancreatic duct, meaning a cell
that is located in the next layer of cells after the duct in
contact with the pancreatic tissue (i.e. not facing the lumen of
the duct.) Thus, in embodiments in which the cell type that gives
rise to a pancreatic organoid is not a ductal cell, the pancreatic
organoid will not express K7 or K19. However, such a pancreatic
organoid will still preferably express one or more general stem
cell progenitor markers such as Sox9.
[0413] A pancreatic organoid of the invention preferably comprises
.alpha. cells, .beta. cells, .delta. cells, and PP cells. In a
further preferred embodiment, a pancreatic organoid comprises
beta-cells. For example, a pancreatic organoid may comprise more
than 1%, more than 5%, more than 10%, more than 15%, or more than
20% beta-cells. Expression of insulin may be used as a marker for
beta cells.
[0414] In an alternative embodiment, the pancreatic organoid
comprises progenitor cell types, optionally with a ductal origin,
that can give rise to differentiated cell-types upon
transplantation into a human or animal. In a preferred embodiment,
the progenitor cell types can give rise to insulin-secreting
beta-cells upon transplantation into a human or animal. The
inventors have shown that human pancreatic organoids, grown
according to the media and methods of the invention, can be
transplanted into mice and stimulate insulin-secreting cells within
one month (see example 4). It can be easily understood that this
could lead to revolutionary treatments for patients with diabetes
and insulin-deficiencies.
[0415] In some embodiments, a pancreatic organoid of the invention
may comprise ductal cells, acinar cells and endocrine cells. In
some embodiments, K19 is used as a marker for ductal cells.
[0416] In some embodiments, a beta-cell exists within pancreatic
islands or Islets of Langerhans. An islet generally comprises
around 1500 cells in vivo, for example, 1300-1700 cells. In one
embodiment, a pancreatic organoid comprises at least 0.5%, at least
1%, at least 1.5%, at least 2%, at least 3%, at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30% or more
Islets of Langerhans by mass. In some embodiments, the Islets of
Langerhans of the pancreatic organoid are composed of approximately
65 to 90% beta cells, approximately 15 to 20% alpha-cells,
approximately 3 to 10% delta cells, and approximately 1% PP cells.
However, this is by no means exclusive. For example, in some
embodiments, it is desirable to have many beta cells in an organoid
of the invention. Alternatively, an organoid may comprise
progenitor cells that may be transplanted so that they
differentiate in vivo.
[0417] In some embodiments, a pancreatic organoid expresses one,
two or all three of Pdx1, Nkx2.2 and Nkx6.1. A pancreatic organoid
may express one, two, three or all four of NeuroD, Pax6, Pax4 and
Mafa. Pax4 serves as a marker for the presence of insulin producing
cells because it is an essential transcription factor for the
differentiation of insulin producing cells from endocrine
progenitor cells during embryonic development. A pancreatic
organoid may express Ngn3.
[0418] In some embodiments, at least one (for example 1, 2, 3, 4,
5) of the following markers can be detected in a pancreatic
organoid of the invention: insulin (ins1 and/or ins2), glucagon
(Gcg), somatostatin, Pdx1 and NeuroD. In some embodiments, at least
one (for example 1, 2, 3, 4, 5) of the following markers can be
detected in a pancreatic organoid of the invention: insulin (ins1
and/or ins2), glucagon (Gcg), somatostatin, Pdx1 and NeuroD and the
following markers are not detected: ptf1a, amy2a4, Pnlip and cela1.
In some embodiments, at least one (for example 1, 2, 3, 4, 5, 6, 7,
8 or 9) of the following markers can be detected in a pancreatic
organoid of the invention: Ptf1a, pancreatic amylase (Amy2a4),
pancreatic lipase (Pnlip), insulin (ins1 and/or ins2), glucagon
(Gcg), somatostatin, chymotrypsin (cela1), Pdx1 and NeuroD.
[0419] In some embodiments, the pancreatic organoids show natural
expression of Lgr5. In some embodiments, the pancreatic organoids
show natural expression of at least Lgr5 and one or more (e.g. 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20) stem cell markers selected from the group consisting of: CK19,
Nestin, CXCR4.sup.+, CD133.sup.+, DCAMKL-1, CD44, Sord, Sox9, CD44,
Prss23, Sp5, Hnf1.alpha., Hnf4a, Sox9, KRT7 and KRT19, prom1,
Cd24a, Lgr4, epcam. Alternatively or additionally, in some
embodiments, pancreatic organoids may be characterised by natural
expression of one or more (for example 1, 2, 3 or 4) of: CK19,
Nestin, (insulin, glucagon) and CXCR4.sup.+.
[0420] In some embodiments, the pancreatic organoids or cells of
the invention express Somatostatin. Somatostatin is a hormone
expressed in differentiated delta cells and so may serve as a
marker for delta cells.
[0421] Alternatively or additionally, in some embodiments,
pancreatic organoids show natural expression of one or more early
endocrine markers, for example at least one or more (e.g. 1, 2, 3,
4, 5, 6 or 7) of the following early endocrine markers: Sox9,
Hnf1b, Hnf6, Hnf1a, Nkx2.2, Nkx6.1 and Pdx1.
[0422] Alternatively or additionally, in some embodiments,
pancreatic organoids show natural expression of one or more early
endocrine markers, for example at least one or more (e.g. 1, 2, 3
or 4) of the following endocrine markers: Foxa2, Hnf6, Hnf1b and
Sox9. In some embodiments, although the pancreatic organoids show
natural expression of one or more (e.g. 1, 2, 3 or 4) of the
following endocrine markers: Foxa2, Hnf6, Hnf1b and Sox9, they do
not show expression of Ngn3.
[0423] Alternatively or additionally, in some embodiments,
pancreatic organoids show natural expression of one or more ductal
markers, for example, one or both of keratin 7 and keratin 19. In
some embodiments, the pancreatic organoids show natural expression
of one or more ductal markers at a significant or detectable level.
Thus, in some embodiments, the pancreatic organoids have a ductal
phenotype. In some embodiments, pancreatic organoids show
expression of one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15) or all of the following markers, selected
from the group: Hnf1A, Hnf1B, Hnf4A, HHEX, ONECUT1, ONECUT2, CDH1,
FOXA2, GATA6, CEBPB, CEBPD, CEBPG, Glul, Krt7, Krt19 and MET.
[0424] However, the pancreatic organoids may have some ductal
features in combination with features of insulin-producing
precursor cells. For example, they may express one or more ductal
markers as shown in FIG. 16B. In some embodiments, a pancreatic
organoid exhibits a gene expression profile relative to adult
pancreas or liver organoids approximately as shown in FIG. 16B. For
example, in some embodiments, these genes are upregulated or down
regulated in pancreatic organoids compared to adult pancreas liver
organoids to approximately the same fold ratio as in FIG. 16B, for
example, less than +/-3%, less than +/-5%, less than +/-10%, less
than +/-20%,
[0425] In some embodiments, insulin-positive cells appear from the
ductal lining in the pancreatic organoids.
[0426] In some embodiments, one or more (e.g. 1, 2, 3, 4, 5, 6 or
7), preferably all of the following genes are upregulated in
pancreas organoids compared to liver organoids: Aaas, Rps4y2,
Atp2c2, Akap2, Uts2, Sox17, Agr2. For example, in some embodiments,
these genes are upregulated in pancreatic organoids compared to
liver organoids to approximately the same fold ratio as in FIG. 19,
for example, less than +/-3%, less than +/-5%, less than +/-10%,
less than +/-20%.
[0427] In one embodiment, a pancreatic organoid comprises at least
10.sup.3, at least 10.sup.4, at least 10.sup.5 or more cells in
total. In one embodiment, a pancreatic organoid comprises more than
50%, more than 60%, more than 70% or more than 80% ductal-like
endocrine progenitor cells However, lower percentages of
ductal-like endocrine progenitor cells are also envisaged.
Barrett's Esophagus (BE) Organoids
[0428] A BE organoid of the invention is Ki67+.
[0429] Preferably a BE organoid has a minimal number (e.g. less
than 25%, less than 20%, less than 10%, less than 5%, less than 2%,
less than 1% cells) of PAS+ and Mucin+ cells 4 days after
withdrawal of Nicotinamide and SB202190 from the expansion medium
to covert it to the differentiation medium.
[0430] In some embodiments, a BE organoid comprises goblet cells.
These may be induced by treatment of the differentiation medium
with a gamma-secretase inhibitor such as DBZ (e.g. at 10 uM), for
example, for 4 days.
[0431] In some embodiments, a Barrett's Esophagus organoid of the
invention comprises Paneth cells.
[0432] In some embodiments, a Barrett's Esophagus organoid of the
invention expresses lysozyme.
Gastric Organoids
[0433] In some embodiments, the gastric organoids of the invention
show natural expression of Lgr5. In some embodiments, gastric
organoids of the invention show natural expression of at least Lgr5
and one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16 or 17) of stem cell markers from the group consisting
of: CK19, Nestin, Somatostatin, CXCR4.sup.+, CD133.sup.+, DCAMKL-1,
CD44, Sord, Sox9, CD44, Prss23, Spy, Hnf1.alpha., Hnf4a, Sox9, KRT7
and KRT19. Alternatively or additionally, in some embodiments
gastric organoids may be characterised by natural expression of one
or more (for example 1, 2 or 3) of: CD133.sup.+, DCAMKL-1 and CD44.
Alternatively or additionally, gastric organoids may be
characterised by CD44 and Sox9.
Prostate Organoids
[0434] In some embodiments, the prostate organoids of the
invention, such as mouse prostates, show natural expression of
Lgr5. In some embodiments, the prostate organoids show natural
expression of luminal prostate markers, such as Cytokeratin 18
(CK18) and Cytokeratin 8 (CK8). In some embodiments, the prostate
organoids of the invention show natural expression of Androgen
Receptor (AR). In some embodiments, the prostate organoids express
basal markers, such as p63 and/or Cytokeratin 5 (CK5). In some
embodiments, when testosterone (e.g. DHT) is added to the medium,
the expression of basal markers, Lgr5 and Tnfrsf19 are
downregulated compared to organoids grown in the absence of
testosterone (e.g. DHT). The prostate specific transcription factor
NKX3.1 is expressed in all conditions. Therefore, in some
embodiments the prostate organoids of the invention show natural
expression of the prostate specific marker Nkx3.1.
[0435] In some embodiments, the prostate organoids of the
invention, for example normal or cancer human prostate organoids,
show natural expression of luminal markers, such as CK18, CK8
and/or B-MSP. In some embodiments, the prostate organoids show
natural expression of AR. In some embodiments, basal epithelial
markers, such as CK14, CK5 and/or p63 are expressed. In some
embodiments, TNFRSF19 is expressed. The prostate specific
transcription factor NKX3.1 is expressed in all conditions.
Therefore, in some embodiments the prostate organoids of the
invention show natural expression of the prostate specific marker
Nkx3.1.
[0436] The addition of testosterone (e.g. DHT) to a culture medium
according to the invention allows prostate organoids to grow that
maintain a stem cell population allowing up to 3-fold faster growth
(than without testosterone) and most (if not all) differentiated
cell types of the prostate (both basal and luminal cells) are also
present. These conditions allow unlimited cell expansion (so far 9
months at 2.5 population doublings a week). Therefore, in some
embodiments, a prostate organoid comprises all differentiated cell
types of the prostate, for example both basal and luminal cells. In
a preferred embodiment, a prostate organoid comprises all
differentiated cell types, for example both basal and luminal
cells, and stem cells.
[0437] In normal tissue, addition of testosterone (e.g. DHT)
increases AR expression in all culture conditions. In some
embodiments, prostate organoids have upregulated AR expression
compared to prostate cells grown in the absence of testosterone. In
tumour tissue AR expression is not influenced by testosterone (e.g.
DHT) addition. Therefore, in some embodiments, a prostate cancer
organoid does not have increased AR expression relative to in vivo
prostate cancer cells. The stem cell marker LGR5 is expressed under
ENRF conditions in prostate organoids from normal tissue. In
prostate organoids obtained from tumour tissue, LGR5 expression is
induced with the addition of testosterone (e.g. DHT). In some
embodiments, prostate organoids express LGR5.
Organoid Functions
[0438] In some embodiments, organoids generated by media and
methods of the present invention, mimic in vivo cell fate decisions
in response to external factors. Preferably, cells and organoids
generated according to the invention also possess tissue-specific
functions.
Pancreatic Organoids
[0439] A pancreatic organoid preferably possesses endocrine and
exocrine pancreatic functions, such as expressing one or more (for
example 1, 2 or all 3) of insulin, glucagon and somatostatin. The
expression of these hormones is tightly regulated by a set of
endocrine pancreas-specific transcription factors, the most
important being Pdx1 and NeuroD. The exocrine pancreas is formed by
acinar and ductal compartments responsible of producing the
digestive enzymes amylase, pancreatic lipase and chymotrypsin,
among others. The expression of these genes is also regulated by
specific exocrine pancreatic genes as Ptf1a.
[0440] Pancreatic cells and organoids according to the present
invention may preferably be capable of secreting insulin, for
example, at a rate of between approximately 1 .mu.g per hour per
10.sup.6 cells and 10 .mu.g per hour per 10.sup.6 cells, for
example, between 2 .mu.g and 6 .mu.g per hour per 10.sup.6 cells.
The level of insulin secretion can be detected by methods well
known in the art, for example, by Western Blot compared to a
reference or by C-peptide Elisa. The preferred method to
demonstrate that pancreatic organoids can secrete insulin is by
testing productin of C-peptide. Proinsulin C-peptide serves as an
important linker between the A- and the B-chains of insulin and
facilitates the efficient assembly, folding, and processing of
insulin in the endoplasmic reticulum. Equimolar amounts of
C-peptide and insulin are then stored in secretory granules of the
pancreatic beta cells and both are eventually released to the
portal circulation. Thus, C-peptide is a preferred marker of
insulin secretion.
[0441] Thus, in one embodiment there is provided a pancreatic
organoid that secretes insulin following transplantation in vivo.
In some embodiments, following transplantation in vivo, the
pancreatic organoid secretes insulin at a rate of at least 1 .mu.g
per hour per 10.sup.6 cells, for example, at least 2 .mu.g per hour
per 10.sup.6 cells, at least 4 .mu.g per hour per 10.sup.6 cells,
at least 6 .mu.g per hour per 10.sup.6 cells, at least 8 .mu.g per
hour per 10.sup.6 cells or at least 10 .mu.g per hour per 10.sup.6
cells, In some embodiments, the cells in the pancreatic organoid
are not capable of secreting insulin and/or do not express insulin
as a marker when cultured in vitro. However, cells from a
pancreatic organoid of the present invention are preferably capable
of secreting insulin in vivo when transplanted into a patient, for
example, into the patient's pancreas. In some embodiments, the
ability to secrete insulin may not be present immediately upon
transplantation, but is present by about one month after
transplantation, for example, by 6 weeks, 2 months or 3 months
after transplantation.
[0442] If an enriched endocrine cell sample is obtained from a
pancreatic organoid of the invention, in some embodiments, 75-85%
of the cells in the enriched endocrine cell sample would be
insulin-secreting cells.
[0443] In some embodiments, the invention provides pancreatic
organoids for use in treating diabetes. In some embodiments the
pancreatic organoids are expanding organoids, whereas in other
embodiments they may be differentiated organoids. In some
embodiments one or more (e.g. 1, 2, 3, 4, 5, 6, 7 etc) whole
organoids are transplanted into an animal or patient, whereas in
other embodiments a sample of cells is transplanted into a
patient.
Crypt-Villus Organoids
[0444] A crypt-villus organoid preferably possesses secretory and
self-renewal functions. For example, a crypt-villus organoid
preferably secretes mucin, enzymatic and hormonal secretions, such
as lysozyme, cholecystokinin, secretin and gastric inhibitory
peptide, and other glycoproteins.
Gastric Organoids
[0445] The human stomach is anatomically and functionally divided
into two major regions. The pyloric antrum close to the intestine
mainly produces protective mucus and secretes hormones such as
gastrin. The gastric corpus secretes hydrochloric acid and gastric
enzymes such as pepsinogen. The gastric epithelium of the both
regions is organized in invaginations called glands. These glands
harbor the gastric stem cells, progenitor cells and differentiated
cells. The precise composition of the differentiated cells varies
according to the function of the anatomic region. In the pyloric
antrum, glands are mainly composed of mucin 6 producing cells and
hormone producing endocrine cells. In the corpus,
pepsinogen-producing chief cells and acid-secreting parietal cells
are dispersed between the mucus producing cells and sparse
endocrine cells. The surface region between gastric glands is
occupied by mucus producing cells that mainly produce the surface
mucin 5.
[0446] Gastric organoids resemble the gastric epithelium in
structure and function. Although they are mostly spheric, they can
have domains with invaginations that most likely resemble glandular
structures. Staining of mucins and pepsinogen shows that the most
abundant cell types in the gastric organoids are mucin 6 producing
mucus cells and pepsinogen producing chief cells (and/or their
progenitors). Accordingly, RT-PCRs indicate the expression of
pepsinogen and mucin 6. Further, expression of gastrin indicates
the presence of endocrine cells and the expression of Lgr5
indicates the presence of stem cells.
[0447] In some embodiments, gastric organoids have natural
expression of one or more (e.g. 1, 2, 3 or 4) of gastrin,
pepsinogen, mucin 6 and/or Lgr5. In some embodiments, gastric
organoids comprise mucin 6 producing mucus cells and pepsinogen
producing chief cells and optionally Lgr5+ stem cells. In some
embodiments, gastric organoids comprise endocrine cells. In some
embodiments, gastric organoids are mostly spheric but have domains
with invaginations that resemble glandular structures.
Prostate Organoids
[0448] In some embodiments, prostatic organoids comprise or consist
of two distinct epithelial lineages, basal cells and luminal cells.
In some embodiments basal cells and luminal cells secrete prostatic
fluids.
[0449] In vivo the prostatic epithelium is strongly folded,
ensuring maximum surface area. The two epithelial lineages form a
simple stratified epithelium with the basal epithelial cells
forming the basal/outer layer and the strongly polarized luminal
epithelial cells situated on top forming the inner/luminal layer.
The luminal compartment is essential for the secretory function of
the prostate. The prostatic fluid is alkaline and is composed of
several proteins, such as Prostate Specific Antigen (PSA), Human
Kallikrein 2 (KLK2) and .beta.-microseminoprotein (.beta.-MSP). The
primary functions of prostatice fluid are: 1) preparing the milieu
of the uterus for the semen, which is performed by the alkalinity
of the fluid and the paracrine functions of .beta.-MSP, and 2)
increasing the fluidity of the seminal fluid, allowing the
spermatozoa to swim freely, which is performed by the proteases PSA
and KLK2 which breakdown seminogelins.
[0450] The expression of secretory proteins is tightly regulated by
the androgen receptor (AR), which binds to testosterone and
subsequently translocates to the nucleus and activates
transcription. Disruptions in AR function show a strong
downregulated of secretory proteins on a transcriptional and
protein level.
[0451] FIG. 43 shows the stratification of the prostate organoids
grown under ENR+Dihydrotestosterone (DHT) conditions, clearly
showing Cytokeratin 5+ basal cells forming an outer layer of cells
and the Cytokeratin 8+ luminal cells forming a strongly polarized
inner layer. In some embodiments, a prostate organoid comprises
cytokeratin 5+ basal cells and cytokeratin 8+ luminal cells,
optionally wherein the Cytokeratin 5+ basal cells forming an outer
layer of cells and the Cytokeratin 8+ luminal cells forming a
strongly polarized inner layer. In some embodiments, a prostate
organoid comprises folded layers of cells, optionally comprising
strong folding. Such folding maximizes the surface area of
secretory cells, showing that on a morphological level prostate
organoids resemble the in vivo prostate. In some embodiments, the
morphology of a prostate organoid resembles the in vivo morphology
of the prostate.
[0452] The prostate organoids cultured in ENR conditions do not any
secrete prostatic fluid into the lumen. By contrast, addition of
testosterone (e.g. DHT) to the medium results in secretion of
fluids, for example prostatic fluid, in the organoid lumen. This is
due to the activation of the AR-dependent transcriptional program
in prostatic organoids by testosterone (e.g. DHT), which results in
secretion of fluids by CK8+ luminals cells. The data show that
prostatic organoids both morphologicaly and functionally resemble
the in vivo prostatic epithelium.
[0453] Accordingly, in some embodiments, for example wherein the
prostate organoids are cultured in a culture medium comprising
testosterone (e.g. DHT), a prostate organoid secretes fluid, for
example prostatic fluid into the lumen of the organoid. In some
embodiments, for example wherein the prostate organoids are
cultured in a culture medium comprising testosterone (e.g. DHT),
the functionality of a prostate organoid resembles the in vivo
functionality of the prostate.
Tissue Fragments
[0454] Within the context of the invention, a tissue fragment is a
part of an adult tissue, preferably a human adult tissue, such as
part of a human adult small intestine, colon or pancreas. Further
examples of human adult tissue in the context of this invention
include stomach, liver and prostate. The tissue may be normal
(healthy) tissue or it may be diseased or infected tissue.
Preferably an organoid as identified herein is therefore not a
tissue fragment. An organoid is preferably obtained using a cell
from an adult tissue, preferably an epithelial stem cell from an
adult tissue, optionally from an adult tissue fragment, more
preferably an epithelial stem cell from an adult tissue or adult
tissue fragment expressing Lgr5. Therefore, within the context of
this invention, a tissue fragment preferably comprises Lgr5+ stem
cells.
[0455] In an embodiment, an organoid is an organoid which is still
being cultured using a method of the invention (preferably using a
culture medium of the invention) and is therefore in contact with
an extracellular matrix. Preferably, an organoid is embedded in a
non-mesenchymal extracellular matrix. Within the context of the
invention, "in contact" means a physical or mechanical or chemical
contact, which means that for separating said organoid from said
extracellular matrix a force needs to be used. In some embodiments,
the extracellular matrix is a gelatinous protein mixture secreted
by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, such as
Matrigel (BD Biosciences). In other embodiments of the invention,
organoids may be removed from culture and used for transplantation
or regenerative purposes. Thus the invention provides an organoid
of the invention for use in transplantation into a mammal,
preferably into a human.
Survival Rate
[0456] The inventors show here, for the first time, that addition
of an inhibitor of ALK4, ALK5, ALK7 or p38 kinase, to the
previously described stem cell culture medium, improved culture
plating efficiency by at least 50% and by more than 100% in some
cases (see table 2). The inventors have also shown that including
both inhibitors (an ALK inhibitor and a p38 inhibitor e.g. A83-01
and SB-202190) in the culture medium synergistically prolongs the
culture period.
[0457] Accordingly, in one embodiment of the invention, the stem
cells survive for at least 3 months, preferably at least 4 months,
at least 5 months, at least 6 months, at least 7 months, at least 9
months, or at least 12 months or more.
Speed of Proliferation
[0458] The speed of proliferation may be assessed in terms of the
cell population doubling level. The population doubling level
refers to the total number of times the cells in the population
have doubled since their primary isolation in vitro. The population
doubling level can be determined by cell counting. Alternatively,
the speed of proliferation can be assessed by a cellular
proliferation assay, for example in which specific fluorescent
probes measure DNA synthesis activity by BrdU incorporation and
cell proliferation state by Ki67 expression (Thermo Scientific*
Cellomics, Millipore).
[0459] Further examples of cellular proliferation assays for stem
cells are readily available can be found online or in journals such
as Current Protocols. One example of many is:
http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catDispl-
ayStyle&catKey=10 1&filterDispName=Cellular Proliferation
Assays for Stem
Cells&filterType=1&OP=filter&filter=ft.sub.--1101%2Ff494303*&bcs_=H4sIAAA-
AAAAAAH2 NsQrDMAxEvOZTsEkdKFmzZC70C4IjakFsGVuOfz
%2FK016F4x284c48YJxfhffin%0ApQ7gn
sMby0ke6x8fRDJMC7hV03u31E6Swh9M1nNUWU1Qq1UFJXgeIvvotnSbn6Lbl1
yPshvQpyq %0ADRIPfQE33R1nKQ21Lvuq17CrAAAA.
[0460] The inventors have observed that using the culture media of
the invention cells can expand by up to an average of 5 times a
week. For example, growing a single cell for two weeks would give
approximately 25 cells on average. The skilled person will
understand that the average population doubling time of the stem
cells of the invention may vary according to several factors, such
as passage number, culture conditions, seeding density etc.
[0461] In one embodiment, the average population doubling time may
be 6 to 48 hours, 12 to 36 hours, 18 to 30 hours, or approximately
24 hours. For example, a stem cell population cultured using a
culture medium of the invention may be expected to double
approximately 4-7 times, or approximately 5 times per week.
[0462] In another embodiment, the average population doubling time
is 12 to 96 hours, 24 to 72 hours, or approximately 72 hours. In
another embodiment, the cell population doubles on average more
than once, more than twice, more than three times, more than four
times or more than five times a week.
Other Properties of Organoids of the Invention
[0463] In a preferred embodiment, an organoid could be cultured
during at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or 1, 2, 3, 4, 5,
6 months or longer. In a preferred embodiment, an organoid could be
cultured during at least 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer. In another
preferred embodiment, an organoid originates from a single cell,
preferably expressing Lgr5, more preferably wherein the single cell
comprises a nucleic acid construct comprising a nucleic acid
molecule of interest.
[0464] The invention further provides an organoid, preferably
comprising at least 50% viable cells, more preferred at least 60%
viable cells, more preferred at least 70% viable cells, more
preferred at least 80% viable cells, more preferred at least 90%
viable cells. Viability of cells may be assessed using Hoechst
staining or Propidium Iodide staining in FACS.
[0465] The viable cells preferably possess tissue-specific
functions, or characteristics of tissue-specific functions, as
described above.
[0466] The inventors have also shown that organoids generated by
media and methods of the present invention can be frozen and stored
at -80.degree. C. or below, such as in liquid nitrogen. Frozen
organoids can be thawed and put into culture without losing their
3D structure and integrity and without significant cell death.
Therefore, in one embodiment, the invention provides frozen
organoids stored at below -5.degree. C., below -10.degree. C.,
below -20.degree. C., below -40.degree. C., below -60.degree. C.,
or below -80.degree. C.
[0467] The cells and organoids of the present invention differ from
any cells and organoids that have been made previously
(WO2009/022907 and WO2010/016766) in that they have better
phenotypic (better differentiation profile including goblet cell
conversion upon addition of gamma secretase inhibitors for the
crypt-villus organoids) and karyotypic integrity, as determined by
the methods outlined above, better survival rates and faster speeds
of cellular proliferation. Accordingly, for intestinal, colon and
pancreatic embodiments, an organoid of the present invention
clearly represents the human intestinal, colon or pancreas
epithelium, with full preservation of phenotypic and karyotypic
integrity and maintenance of proliferation and differentiation.
Uses of Stem Cells or Organoids of the Invention
[0468] The invention provides the use of an organoid or expanded
population of cells of the invention for use in drug screening,
(drug) target validation, (drug) target discovery, toxicology and
toxicology screens, personalized medicine, regenerative medicine
and/or as ex vivo cell/organ models, such as disease models.
[0469] Cells and organoids cultured according to the media and
methods of the invention are thought to faithfully represent the in
vivo situation. This is true both for expanded populations of cells
and organoids grown from normal tissue and for expanded populations
of cells and organoids grown from diseased tissue. Therefore, as
well as providing normal ex vivo cell/organ models, the organoids
or expanded population of cells of the invention can be used as ex
vivo disease models.
[0470] Organoids of the invention can also be used for culturing of
a pathogen and thus can be used as ex vivo infection models.
Examples of pathogens that may be cultured using an organoid of the
invention include viruses, bacteria, prions or fungi that cause
disease in its animal host. Thus an organoid of the invention can
be used as a disease model that represents an infected state. In
some embodiments of the invention, the organoids can be used in
vaccine development and/or production.
[0471] Diseases that can be studied by the organoids of the
invention thus include genetic diseases, metabolic diseases,
pathogenic diseases, inflammatory diseases etc, for example
including, but not limited to: cystic fibrosis, inflammatory bowel
disease (such as Crohn's disease), carcinoma, adenoma,
adenocarcinoma, colon cancer, diabetes (such as type I or type II),
Barrett's esophagus, Gaucher's disease, alpha-1-antitrypsin
deficiency, Lesch-Nyhan syndrome, anaemia,
Schwachman-Bodian-Diamond syndrome, polycythaemia vera, primary
myelofibrosis, glycogen storage disease, familial
hypercholestrolaemia, Crigler-Najjar syndrome, hereditary
tyrosinanaemia, Pompe disease, progressive familial cholestasis,
Hreler syndrome, SCID or leaky SCID, Omenn syndrome, Cartilage-hair
hypoplasia, Herpes simplex encephalitis, Scleroderma, Osteogenesis
imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy,
Dyskeratosis congenitor etc.
[0472] Traditionally, cell lines and more recently iPS cells have
been used as ex vivo cell/organ and/or disease models (for example,
see Robinton et al. Nature 481, 295, 2012). However, these methods
suffer a number of challenges and disadvantages. For example, cell
lines cannot be obtained from all patients (only certain biopsies
result in successful cell lines) and therefore, cell lines cannot
be used in personalised diagnostics and medicine. iPS cells usually
require some level of genetic manipulation to reprogramme the cells
into specific cell fates. Alternatively, they are subject to
culture conditions that affect karotypic integrity and so the time
in culture must be kept to a minimum (this is also the case for
human embryonic stem cells). This means that iPS cells cannot
accurately represent the in vivo situation but instead are an
attempt to mimic the behaviour of in vivo cells. Cell lines and iPS
cells also suffer from genetic instability.
[0473] By contrast, the organoids of the invention provide a
genetically stable platform which faithfully represents the in vivo
situation. The organoids of the invention can also be expanded
continuously, providing a good source of genetically stable cells.
In particular, an expanding population can be "split", meaning that
the organoid is split apart and all cells of the organoid are
divided into new culture dishes or flasks. The divided cells are
removed from the organoid and can then themselves be cultured and
expanded to produce new organoids containing further expanded
populations that can then be split again. Splits are also referred
to herein as "passages". An organoid of the invention may be
cultured for 1 or more passages, for example, 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25, 30 or more passages, for example, 20-30
passages, 30-35 passages, 32-40 passages or more. In some
embodiments, an expanding cell population or organoid is split once
a month, once every two weeks, once a week, twice a week, three
times a week, four times a week, five times a week, six times a
week or daily. Thus the organoids of the invention can provide an
ongoing source of genetically stable cellular material. In some
embodiments, the expanding organoids of the invention comprise all
differentiated cell types that are present in the corresponding in
vivo situation. In other embodiments, the organoids of the
invention may be differentiated to provide all differentiated cell
types that are present in vivo. Thus the organoids of the invention
can be used to gain mechanistic insight into a variety of diseases
and therapeutics, to carry out in vitro drug screening, to evaluate
potential therapeutics, to identify possible targets (e.g.
proteins) for future novel (drug) therapy development and/or to
explore gene repair coupled with cell-replacement therapy.
[0474] The organoids of the invention can be frozen and thawed and
put into culture without losing their genetic integrity or
phenotypic characteristics and without loss of proliferative
capacity. Thus the organoids can be easily stored and
transported.
[0475] For these reason the organoids or expanded populations of
cells of the invention can be a tool for drug screening, target
validation, target discovery, toxicology and toxicology screens and
personalized medicine.
[0476] Accordingly, in a further aspect, the invention provides the
use of the expanded stem cell population or organoid, such as
intestinal crypt-villus organoids or pancreatic organoids according
to the invention in a drug discovery screen, toxicity assay or in
medicine, such as regenerative medicine. For example, any one of
the small intestinal, colon, pancreatic, gastric, liver or prostate
organoids may be used in a drug discovery screen, toxicity assay or
in medicine, such as regenerative medicine.
Mucosal Vaccines
[0477] An additional important use of the organoids is in the
development of mucosal vaccinations. Mucosal vaccines are vaccines
that are administered via the mucosa. This can be any mucosal
surface such as via the nose, mouth, or rectum. They can be
administered via an inhaler, a spray or other external aids. This
has several clear benefits over injections such as that no medical
staff are needed for administering the vaccine, which may be
important, for example in developing countries.
[0478] In the intestine, M cells (or "microfold cells") are cells
found in the follicle-associated epithelium of the aggregated
lymphoid nodules of the ileum. They transport organisms and
particles from the gut lumen to immune cells across the epithelial
barrier, and thus are important in stimulating mucosal immunity
They have the unique ability to take up antigen from the lumen of
the small intestine via endocytosis or phagocytosis, and then
deliver it via transcytosis to dendritic cells (an antigen
presenting cell) and lymphocytes (namely T cells) located in a
unique pocket-like structure on their basolateral side.
[0479] FIG. 48 shows that mouse organoids can develop into M cells
when stimulated with RANK ligand. FIG. 49 shows that it is also
possible to generate M cells in human intestinal organoids.
Therefore, in some embodiments of the invention, the expanded cell
population comprises M cells. In some embodiments of the invention,
an organoid, for example a small-intestinal organoid, comprises M
cells.
[0480] The efficiency of mucosal vaccines can be substantially
increased when they are targeted to M cells. Therefore, the
expanded stem cell population or organoid of the invention can be
used for testing the ability of M cells to take up pathogens or
antigens and to present them to the immune system. Therefore, in
some embodiments the invention provides the use of the expanded
stem cell population or organoid of the invention in drug
screening, for example in vaccine development and/or vaccine
production. For example, in some embodiments the expanded stem cell
population or organoid may be used for the development or
production of vaccines against viral, bacterial, fungal or other
parasitic infections, for example (but not limited to) cholera,
Respiratory syncytial virus (RSV), Rotavirus and HIV. In a
particular embodiment, the invention provides small intestinal
organoids that have been differentiated in a culture medium of the
invention comprising RANKL, for use in mucosal vaccine
development.
Drug Screening
[0481] For preferably high-throughput purposes, said expanded stem
cell population or organoid of the invention, such as crypt-villus
organoids or pancreatic organoids, are cultured in multiwell plates
such as, for example, 96 well plates or 384 well plates. Libraries
of molecules are used to identify a molecule that affects said
organoids. Preferred libraries comprise antibody fragment
libraries, peptide phage display libraries, peptide libraries (e.g.
LOPAP.TM., Sigma Aldrich), lipid libraries (BioMol), synthetic
compound libraries (e.g. LOP AC.TM., Sigma Aldrich) or natural
compound libraries (Specs, TimTec). Furthermore, genetic libraries
can be used that induce or repress the expression of one of more
genes in the progeny of the stem cells. These genetic libraries
comprise cDNA libraries, antisense libraries, and siRNA or other
non-coding RNA libraries. The cells are preferably exposed to
multiple concentrations of a test agent for a certain period of
time. At the end of the exposure period, the cultures are
evaluated. The term "affecting" is used to cover any change in a
cell, including, but not limited to, a reduction in, or loss of,
proliferation, a morphological change, and cell death. Said
expanded stem cell population or organoid of the invention such as
crypt-villus organoids or pancreatic organoids can also be used to
identify drugs that specifically target epithelial carcinoma cells,
but not said expanded stem cell population or organoid of the
invention, such as crypt-villus organoids or pancreatic
organoids.
[0482] The inventors have shown that it is possible to take a
biopsy from the small intestine and expand it for just 7-14 days
and obtain an organoid which is ready for carrying out a drug
screen. The ability to obtain a useful organoid of the invention in
such a short time shows that the organoids would be highly useful
for testing individual patient responses to specific drugs and
tailoring treatment according to the responsiveness. In some
embodiments, wherein the organoid is obtained from a biopsy from a
patient, the organoid is cultured for less than 21 days, for
example less than 14 days, less than 13 days, less than 12 days,
less than 11 days, less than 10 days, less than 9 days, less than 8
days, less than 7 days (etc).
[0483] The organoids are also useful for wider drug discovery
purposes. For example, FIGS. 32 to 40 show that small intestinal
organoids taken from healthy patients and from cystic fibrosis
patients can be used to test drugs against cystic fibrosis.
Specifically, FIGS. 32 to 40 show that forskolin-induced swelling
of normal small intestinal organoids is dependent upon the cystic
fibrosis transmembrane conductance regulator (CFTR), and thus it is
possible to test for correction of CFTR function using
forskolin-induced swelling as a positive read out. Therefore, in
some embodiments, the organoids of the invention could be used for
screening for cystic fibrosis drugs. However, it will be understood
by the skilled person that the organoids of the invention would be
widely applicable as drug screening tools for infectious,
inflammatory and neoplastic pathologies of the human
gastrointestinal tract and other diseases of the gastrointestinal
tract and infectious, inflammatory and neoplastic pathologies and
other diseases of other tissues described herein including
pancreas, liver and prostate. In some embodiments the organoids of
the invention could be used for screening for cancer drugs.
[0484] In some embodiments, the expanded cell populations, for
example the organoids of the invention or organoids obtained using
media and methods of the invention can be used to test libraries of
chemicals, antibodies, natural product (plant extracts), etc for
suitability for use as drugs, cosmetics and/or preventative
medicines. For instance, in some embodiments, a cell biopsy from a
patient of interest, such as tumour cells from a cancer patient,
can be cultured using culture media and methods of the invention
and then treated with a chemical compound or a chemical library. It
is then possible to determine which compounds effectively modify,
kill and/or treat the patient's cells. This allows specific patient
responsiveness to a particular drug to be tested thus allowing
treatment to be tailored to a specific patient. Thus, this allows a
personalized medicine approach.
[0485] The added advantage of using the organoids for identifying
drugs in this way is that it is also possible to screen normal
organoids (organoids derived from healthy tissue) to check which
drugs and compounds have minimal effect on healthy tissue. This
allows screening for drugs with minimal off-target activity or
unwanted side-effects.
[0486] Drugs for any number of diseases can be screened in this
way. For example the organoids of the invention can be used for
screening for drugs for cystic fibrosis, Barrett's esophagus,
carcinomas, adenocarcinomas, adenomas, inflammatory bowel disease
(such as Crohn's disease), liver disease etc. The testing
parameters depend on the disease of interest. For example, when
screening for cancer drugs, cancer cell death is usually the
ultimate aim. For cystic fibrosis, measuring the expansion of the
organoids in response to the drugs and stimuli of CFTR is of
interest. In other embodiments, metabolics or gene expression may
be evaluated to study the effects of compounds and drugs of the
screen on the cells or organoids of interest.
[0487] Therefore, the invention provides a method for screening for
a therapeutic or prophylactic drug or cosmetic, wherein the method
comprises: [0488] culturing an expanded cell population (for
example, an organoid) of the invention, for example with a culture
medium of the invention, optionally for less than 21 days; [0489]
exposing said expanded cell population (for example, an organoid)
of the invention to one or a library of candidate molecules; [0490]
evaluating said expanded cell populations (for example, organoids)
for any effects, for example any change in the cell, such as a
reduction in or loss of proliferation, a morphological change
and/or cell death; [0491] identifying the candidate molecule that
causes said effects as a potential drug or cosmetic.
[0492] In some embodiments, computer- or robot-assisted culturing
and data collection methods are employed to increase the throughput
of the screen.
[0493] In some embodiments, expanded cell population (for example,
an organoid) is obtained from a patient biopsy. In some
embodiments, the candidate molecule that causes a desired effect on
the cultured expanded cell population (for example, an organoid) is
administered to said patient.
[0494] Accordingly, in one aspect, there is provided a method of
treating a patient comprising: [0495] (a) obtaining a biopsy from
the diseased tissue of interest in the patient; [0496] (b)
screening for a suitable drug using a screening method of the
invention; and [0497] (c) treating said patient with the drug
obtained in step (b).
[0498] In some embodiments, the drug or cosmetic is used for
treating, preventing or ameliorating symptoms of genetic diseases,
metabolic diseases, pathogenic diseases, inflammatory diseases etc,
for example including, but not limited to: cystic fibrosis,
inflammatory bowel disease (such as Crohn's disease), carcinoma,
adenoma, adenocarcinoma, colon cancer, diabetes (such as type I or
type II), Barrett's esophagus, Gaucher's diseases,
alpha-1-antitrypsin deficiency, Lesch-Nyhan syndrome, anaemia,
Schwachman-Bodian-Diamond syndrome, polycythaemia vera, primary
myelofibrosis, glycogen storage disease, familial
hypercholestrolaemia, Crigler-Najjar syndrome, hereditary
tyrosinanaemia, Pompe disease, progressive familial cholestasis,
Hreler syndrome, SCID or leaky SCID, Omenn syndrome, Cartilage-hair
hypoplasia, Herpes simplex encephalitis, Scleroderma, Osteogenesis
imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy,
Dyskeratosis congenitor etc.
Target Discovery
[0499] In some embodiments, the organoids of the invention or cells
grown using the culture media and methods of the invention can be
used for target discovery. Cells of the organoids originating from
healthy or diseased tissue may be used for target identification.
The organoids of the invention may be used for discovery of drug
targets for cystic fibrosis, inflammatory bowel disease (such as
Crohn's disease), carcinoma, adenoma, adenocarcinoma, colon cancer,
diabetes (such as type I or type II), Barrett's esophagus Gaucher's
disease, alpha-1-antitrypsin deficiency, Lesch-Nyhan syndrome,
anaemia, Schwachman-Bodian-Diamond syndrome, polycythaemia vera,
primary myelofibrosis, glycogen storage disease, familial
hypercholestrolaemia, Crigler-Najjar syndrome, hereditary
tyrosinanaemia, Pompe disease, progressive familial cholestasis,
Hreler syndrome, SCID or leaky SCID, Omenn syndrome, Cartilage-hair
hypoplasia, Herpes simplex encephalitis, Scleroderma, Osteogenesis
imperfecta, Becker muscular dystrophy, Duchenne muscular dystrophy,
Dyskeratosis congenitor etc. Cells and organoids cultured according
to the media and methods of the invention are thought to faithfully
represent the in vivo situation. For this reason they can be a tool
to find novel (molecular) targets in specific diseases.
[0500] To search for a new drug target, a library of compounds
(such as siRNA) may be used to transduce the cells and inactivate
specific genes. In some embodiments, cells are transduced with
siRNA to inhibit the function of a (large) group of genes. Any
functional read out of the group of genes or specific cellular
function can be used to determine if a target is relevant for the
study. A disease-specific read out can be determined using assays
well known in the art. For example, cellular proliferation is
assayed to test for genes involved in cancer. For example, a
Topflash assay as described herein, may be used to detect changes
in Wnt activity caused by siRNA inhibition. Where growth reduction
or cell death occurs, the corresponding siRNA related genes can be
identified by methods known in the art. These genes are possible
targets for inhibiting growth of these cells. Upon identification,
the specificity of the identified target for the cellular process
that was studied will need to be determined by methods well known
in the art. Using these methods, new molecules can be identified as
possible drug targets for therapy.
Target and Drug Validation Screens
[0501] Patient-specific organoids obtained from diseased and/or
normal tissue can be used for target validation of molecules
identified in high throughput screens. The same goes for the
validation of compounds that were identified as possible
therapeutic drugs in high throughput screens. The use of primary
patient material expanded in the organoid culture system can be
useful to test for false positives, etc from high throughput drug
discovery cell line studies.
[0502] In some embodiments, the expanded stem cell population (for
example, organoid of the invention), such as crypt-villus organoids
or pancreatic organoids can be used for validation of compounds
that have been identified as possible drugs or cosmetics in a
high-throughput screen.
Toxicity Assay
[0503] Said expanded stem cell population (for example, organoid of
the invention), such as crypt-villus organoids or pancreatic
organoids, can further replace the use of cell lines such as Caco-2
cells in toxicity assays of potential novel drugs or of known or
novel food supplements.
[0504] Toxicology screens work in a similar way to drug screens (as
described above) but they test for the toxic effects of drugs and
not therapeutic effects. Therefore, in some embodiments, the
effects of the candidate compounds are toxic.
Culturing Pathogens
[0505] Furthermore, said expanded stem cell population (for
example, organoid of the invention), such as crypt-villus organoids
or pancreatic organoids, can be used for culturing of a pathogen
such as a norovirus which presently lacks a suitable tissue culture
or animal model.
Regenerative Medicine and Transplantation
[0506] Cultures comprising the expanded stem cell population (for
example, organoid of the invention), such as crypt-villus organoids
or pancreatic organoids are useful in regenerative medicine, for
example in post-radiation and/or post-surgery repair of the
intestinal epithelium, in the repair of the intestinal epithelium
in patients suffering from inflammatory bowel disease such as
Crohn's disease and ulcerative colitis, and in the repair of the
intestinal epithelium in patients suffering from short bowel
syndrome. Further use is present in the repair of the intestinal
epithelium in patients with hereditary diseases of the small
intestine/colon. Cultures comprising pancreatic organoids are also
useful in regenerative medicine, for example as implants after
resection of the pancreas or part thereof and for treatment of
diabetes such as diabetes I and diabetes II.
[0507] In an alternative embodiment, the expanded epithelial stem
cells are reprogrammed into related tissue fates such as, for
example, pancreatic cells including pancreatic beta-cells. Thus
far, it has not been possible to regenerate pancreatic cells from
adult stem cells. The culturing methods of the present invention
will enable to analyse for factors that trans-differentiate the
closely related epithelial stem cell to a pancreatic cell,
including a pancreatic beta-cell.
[0508] It will be clear to a skilled person that gene therapy can
additionally be used in a method directed at repairing damaged or
diseased tissue. Use can, for example, be made of an adenoviral or
retroviral gene delivery vehicle to deliver genetic information,
like DNA and/or RNA to stem cells. A skilled person can replace or
repair particular genes targeted in gene therapy. For example, a
normal gene may be inserted into a nonspecific location within the
genome to replace a nonfunctional gene. In another example, an
abnormal gene sequence can be replaced for a normal gene sequence
through homologous recombination. Alternatively, selective reverse
mutation can return a gene to its normal function. A further
example is altering the regulation (the degree to which a gene is
turned on or off) of a particular gene. Preferably, the stem cells
are ex vivo treated by a gene therapy approach and are subsequently
transferred to the mammal, preferably a human being in need of
treatment.
[0509] Since small biopsies taken from adult donors can be expanded
without any apparent limit or genetic harm, the technology may
serve to generate transplantable epithelium for regenerative
purposes. The fact that organoids can be frozen and thawed and put
into culture without losing their 3D structure and integrity and
without significant cell death further adds to the applicability of
organoids for transplantation purposes. Furthermore, in some
embodiments, organoids embedded in, or in contact with, an ECM can
be transplanted into a mammal, preferably into a human. In another
embodiment, organoids and ECM can be transplanted simultaneously
into a mammal, preferably into a human.
[0510] The skilled person would understand that an ECM can be used
as a 3D scaffold for obtaining tissue-like structures comprising
expanded populations of cells or organoids according to the
invention. Such structures can then be transplanted into a patient
by methods well known in the art. An ECM scaffold can be made
synthetically using ECM proteins, such as collagen and/or laminin,
or alternatively an ECM scaffold can be obtained by
"decellularising" an isolated organ or tissue fragment to leave
behind a scaffold consisting of the ECM (for example see
Macchiarini et al. The Lancet, Volume 372, Issue 9655, Pages
2023-2030, 2008). In some embodiments, an ECM scaffold can be
obtained by decellularising an organ or tissue fragment, wherein
optionally said organ or tissue fragment is from the pancreas,
liver, intestine, stomach or prostate.
[0511] As mentioned above, the invention provides an organoid or
population of cells of the invention for use in transplantation
into a mammal, preferably into a human. Also provided is a method
of treating a patient in need of a transplant comprising
transplanting an organoid or population of cells of the invention
into said patient, wherein said patient is a mammal, preferably a
human.
[0512] Advantageously, the invention enables a small biopsy to be
taken from an adult donor and expanded without any apparent limit
or genetic harm and so the technology provided herein may serve to
generate transplantable epithelium for regenerative purposes.
[0513] Significantly, the inventors have found that when human
pancreatic organoids of the invention are transplanted under the
peri-renal capsule in mice, these cells differentiate to form
mature beta cells that secrete insulin. This is significant as it
means that even if the population of cells or organoid of the
invention does not secrete insulin at a detectable level whilst the
cells or organoids are being cultured in vitro, these cells may be
useful for transplantation into a patient for the treatment of an
insulin-deficiency disorder such as diabetes.
[0514] Thus the invention comprises a method of treating an
insulin-deficiency disorder such as diabetes, or a patient having a
dysfunctional pancreas, comprising transplanting a pancreatic
organoid of the invention or cells from a pancreatic organoid of
the invention into the patient.
[0515] In some embodiments, the cells or organoid do not express or
secrete insulin upon transplantation into the patient but
differentiate within the patient such that they secrete insulin.
For example, the ability to secrete insulin may not be detectable
immediately upon transplantation, but may be present by about one
month after transplantation, for example, by 6 weeks, 2 months or 3
months after transplantation.
[0516] The patient is preferably a human, but may alternatively be
a non-human mammal, such as a cat, dog, horse, cow, pig, sheep,
rabbit or mouse.
[0517] Thus, included within the scope of the invention are methods
of treatment of a human or non-human animal patient through
cellular therapy. Such cellular therapy encompasses the application
of the stem cells or organoids of the invention to the patient
through any appropriate means. Specifically, such methods of
treatment involve the regeneration of damaged tissue. In accordance
with the invention, a patient can be treated with allogeneic or
autologous stem cells or organoids. "Autologous" cells are cells
which originated from the same organism into which they are being
re-introduced for cellular therapy, for example in order to permit
tissue regeneration. However, the cells have not necessarily been
isolated from the same tissue as the tissue they are being
introduced into. An autologous cell does not require matching to
the patient in order to overcome the problems of rejection.
"Allogeneic" cells are cells which originated from an individual
which is different from the individual into which the cells are
being introduced for cellular therapy, for example in order to
permit tissue regeneration, although of the same species. Some
degree of patient matching may still be required to prevent the
problems of rejection.
[0518] Generally the cells or organoids of the invention are
introduced into the body of the patient by injection or
implantation. Generally the cells will be directly injected into
the tissue in which they are intended to act. Alternatively, the
cells will be injected through the portal vein. A syringe
containing cells of the invention and a pharmaceutically acceptable
carrier is included within the scope of the invention. A catheter
attached to a syringe containing cells of the invention and a
pharmaceutically acceptable carrier is included within the scope of
the invention.
[0519] The skilled person will be able to select an appropriate
method and route of administration depending on the material that
is being transplanted (i.e. population of cells, single cells in
cell suspension, organoids or fragments of organoids) as well as
the organ that is being treated.
[0520] As discussed above, cells of the invention can be used in
the regeneration of tissue. In order to achieve this function,
cells may be injected or implanted directly into the damaged
tissue, where they may multiply and eventually differentiate into
the required cell type, in accordance with their location in the
body. Alternatively, the organoid can be injected or implanted
directly into the damaged tissue. Tissues that are susceptible to
treatment include all damaged tissues, particularly including those
which may have been damaged by disease, injury, trauma, an
autoimmune reaction, or by a viral or bacterial infection. In some
embodiments of the invention, the cells or organoids of the
invention are used to regenerate the colon, small intestine,
pancreas, esophagus or gastric system.
[0521] For example, in one embodiment, the cells or organoids of
the invention are injected into a patient using a Hamilton
syringe.
[0522] The skilled person will be aware what the appropriate dosage
of cells or organoids of the invention will be for a particular
condition to be treated.
[0523] In one embodiment the cells or organoids of the invention,
either in solution, in microspheres or in microparticles of a
variety of compositions, will be administered into the artery
irrigating the tissue or the part of the damaged organ in need of
regeneration. Generally such administration will be performed using
a catheter. The catheter may be one of the large variety of balloon
catheters used for angioplasty and/or cell delivery or a catheter
designed for the specific purpose of delivering the cells to a
particular local of the body. For certain uses, the cells or
organoids may be encapsulated into microspheres made of a number of
different biodegradable compounds, and with a diameter of about 15
.mu.m. This method may allow intravascularly administered cells or
organoids to remain at the site of damage, and not to go through
the capillary network and into the systemic circulation in the
first passage. The retention at the arterial side of the capillary
network may also facilitate their translocation into the
extravascular space.
[0524] In another embodiment, the cells or organoids may be
retrograde injected into the vascular tree, either through a vein
to deliver them to the whole body or locally into the particular
vein that drains into the tissue or body part to which the cells or
organoids are directed. For this embodiment many of the
preparations described above may be used.
[0525] In another embodiment, the cells or organoids of the
invention may be implanted into the damaged tissue adhered to a
biocompatible implant. Within this embodiment, the cells may be
adhered to the biocompatible implant in vitro, prior to
implantation into the patient. As will be clear to a person skilled
in the art, any one of a number of adherents may be used to adhere
the cells to the implant, prior to implantation. By way of example
only, such adherents may include fibrin, one or more members of the
integrin family, one or more members of the cadherin family, one or
more members of the selectin family, one or more cell adhesion
molecules (CAMs), one or more of the immunoglobulin family and one
or more artificial adherents. This list is provided by way of
illustration only, and is not intended to be limiting. It will be
clear to a person skilled in the art, that any combination of one
or more adherents may be used.
[0526] In another embodiment, the cells or organoids of the
invention may be embedded in a matrix, prior to implantation of the
matrix into the patient. Generally, the matrix will be implanted
into the damaged tissue of the patient. Examples of matrices
include collagen based matrices, fibrin based matrices, laminin
based matrices, fibronectin based matrices and artificial matrices.
This list is provided by way of illustration only, and is not
intended to be limiting.
[0527] In a further embodiment, the cells or organoids of the
invention may be implanted or injected into the patient together
with a matrix forming component. This may allow the cells to form a
matrix following injection or implantation, ensuring that the cells
or organoids remain at the appropriate location within the patient.
Examples of matrix forming components include fibrin glue liquid
alkyl, cyanoacrylate monomers, plasticizers, polysaccharides such
as dextran, ethylene oxide-containing oligomers, block co-polymers
such as poloxamer and Pluronics, non-ionic surfactants such as
Tween and Triton'8', and artificial matrix forming components. This
list is provided by way of illustration only, and is not intended
to be limiting. It will be clear to a person skilled in the art,
that any combination of one or more matrix forming components may
be used.
[0528] In a further embodiment, the cells or organoids of the
invention may be contained within a microsphere. Within this
embodiment, the cells may be encapsulated within the centre of the
microsphere. Also within this embodiment, the cells may be embedded
into the matrix material of the microsphere. The matrix material
may include any suitable biodegradable polymer, including but not
limited to alginates, Poly ethylene glycol (PLGA), and
polyurethanes. This list is provided by way of example only, and is
not intended to be limiting.
[0529] In a further embodiment, the cells or organoids of the
invention may be adhered to a medical device intended for
implantation. Examples of such medical devices include stents,
pins, stitches, splits, pacemakers, prosthetic joints, artificial
skin, and rods. This list is provided by way of illustration only,
and is not intended to be limiting. It will be clear to a person
skilled in the art, that the cells may be adhered to the medical
device by a variety of methods. For example, the cells or organoids
may be adhered to the medical device using fibrin, one or more
members of the integrin family, one or more members of the cadherin
family, one or more members of the selectin family, one or more
cell adhesion molecules (CAMs), one or more of the immunoglobulin
family and one or more artificial adherents. This list is provided
by way of illustration only, and is not intended to be limiting. It
will be clear to a person skilled in the art, that any combination
of one or more adherents may be used.
Methods of the Invention
[0530] The invention also provides a method for expanding a
population of stem cells, wherein the method comprises: [0531] a)
providing a population of stem cells; [0532] b) providing a culture
medium according to the invention; [0533] c) contacting the stem
cells with the culture medium; and [0534] d) culturing the cells
under appropriate conditions.
[0535] Furthermore, the invention provides a method for expanding
isolated tissue fragments, wherein the method comprises: [0536] a)
providing an isolated tissue fragment; [0537] b) providing a
culture medium according to the invention; [0538] c) contacting the
isolated tissue fragment with the culture medium; and [0539] d)
culturing the cells under appropriate conditions.
[0540] A method for `expanding` a population of cells or isolated
tissue fragments is one that involves maintaining or increasing the
number of stem cells in an initial population to generate an
expanded population of stem cells which retain their
undifferentiated phenotype and self-renewing properties. However,
it may also include the production of differentiating progeny,
which may, for example, form tissue-like structures contributing to
organoid formation. Hence, there are herein provided methods for
obtaining an organoid, such as a small intestinal (crypt-villus)
organoid, a colon organoid, a pancreatic organoid, a gastric
organoid, a prostatic organoid, a liver organoid, an adenocarcinoma
organoid, a carcinoma organoid or a Barrett's Esophagus organoid,
comprising culturing stem cells or tissue fragments comprising said
stem cells in a culture medium of the invention. The invention
provides a method for expanding a single stem cell or a population
of stem cells, preferably to generate an organoid, wherein the
method comprises culturing the single stem cell, population of stem
cells or tissue fragment in a culture medium according to the
invention. In some embodiments, the method for obtaining an
organoid comprises culturing the stem cells or tissue fragments
with a first "expansion" medium, followed by culturing the stem
cells or tissue fragments with a second "differentiation"
medium.
[0541] In some embodiments, the differentiation medium does not
comprise certain components of the expansion medium, for example,
the differentiation medium does not comprise Wnt, Rspondin,
nicotinamide, a TGF-beta inhibitor and/or a p38 inhibitor.
[0542] In some embodiments, the method for expanding a single stem
cell or a population of stem cells, preferably to generate an
organoid, comprises expanding the single stem cell, population of
stem cells or tissue fragment in a first culture medium according
to the invention, and optionally, differentiating the expanded
cells or tissue fragments in a second culture medium according to
the invention.
[0543] Thus the invention provides a method for expanding a single
stem cell or a population of stem cells, preferably to generate an
organoid, wherein the method comprises: [0544] providing a stem
cell, a population of stem cells or an isolated tissue fragment;
[0545] providing a culture medium according to the invention;
[0546] contacting the stem cells with the culture medium; [0547]
culturing the cells under appropriate conditions.
[0548] In some embodiments, the method comprises bringing the stem
cell, the population of stem cells or the isolated tissue fragment
and the culture medium into contact with an extracellular matrix or
a 3D matrix that mimics the extracellular matrix by its interaction
with the cellular membrane proteins such as integrins, for example
a laminin-containing extracellular matrix such as Matrigel.TM. (BD
Biosciences). In some embodiments, the culture medium is diffused
into the extracellular matrix.
[0549] In some embodiments, the invention provides a method for
expanding a single stem cell or a population of stem cells or
tissue fragment, preferably to generate an organoid, wherein the
method comprises: [0550] culturing the stem cell, population of
stem cells or tissue fragment in a first expansion medium; [0551]
continuing to culture the stem cell, population of stem cells or
tissue fragment and replenishing the medium with a differentiation
medium, wherein the differentiation medium does not comprise one or
more of, preferably all of the factors selected from: a TGF-beta
inhibitor, a p38 inhibitor, nicotinamide and Wnt.
[0552] In some embodiments, the invention provides a method for
expanding a single stem cell or a population of stem cells,
preferably to generate an organoid of a tissue of interest,
comprising:
[0553] expanding stem cells or tissue fragments from said tissue of
interest in a culture medium of the invention that is suitable for
said tissue of interest; and optionally differentiating the
expanded stem cells or tissue fragments in a culture medium of the
invention that is suitable for said tissue of interest.
[0554] Isolated stem cells are preferably cultured in a
microenvironment that mimics at least in part a cellular niche in
which said stem cells naturally reside. Said cellular niche is
mimicked by culturing said stem cells in the presence of
biomaterials, such as matrices, scaffolds, and culture substrates
that represent key regulatory signals controlling stem cell fate.
Said biomaterials comprise natural, semi-synthetic and synthetic
biomaterials, and/or mixtures thereof. A scaffold provides a
two-dimensional or three dimensional network. Suitable synthetic
materials for said scaffold comprise polymers selected from porous
solids, nanofibers, and hydrogels such as, for example, peptides
including self-assembling peptides, hydrogels composed of
polyethylene glycol phosphate, polyethylene glycol fumarate,
polyacrylamide, polyhydroxyethyl methacrylate, polycellulose
acetate, and/or co-polymers thereof (see, for example, Saha et al,
2007 Curr Opin Chem Biol 1 1(4) 381-387, Saha et al, 2008
Biophysical Journal 95 4426-4438, Little et al, 2008 Chem Rev 108,
1787-1796). As is known to a skilled person, the mechanical
properties such as, for example, the elasticity of the scaffold
influences proliferation, differentiation and migration of stem
cells. A preferred scaffold comprises biodegradable (co)polymers
that are replaced by natural occurring components after
transplantation in a subject, for example to promote tissue
regeneration and/or wound healing. It is furthermore preferred that
said scaffold does not substantially induce an immunogenic response
after transplantation in a subject. Said scaffold is supplemented
with natural, semi-synthetic or synthetic ligands, which provide
the signals that are required for proliferation and/or
differentiation, and/or migration of stem cells. In a preferred
embodiment, said ligands comprise defined amino acid fragments.
Examples of said synthetic polymers comprise Pluronic.RTM. F 127
block copolymer surfactant (BASF), and Ethisorb.RTM. (Johnson and
Johnson).
[0555] A cellular niche is in part determined by the stem cells and
surrounding cells, and the extracellular matrix (ECM) that is
produced by the cells in said niche. In one method of the
invention, isolated crypts or epithelial stem cells are attached to
an ECM. ECM is composed of a variety of polysaccharides (mostly
heparin sulphate proteoglycans), water, elastin, and glycoproteins,
wherein the glycoproteins comprise collagen, entactin (nidogen),
fibronectin, and laminin ECM is secreted by connective tissue
cells. Different types of ECM are known, comprising different
compositions including different types of glycoproteins and/or
different combination of glycoproteins. Said ECM can be provided by
culturing ECM-producing cells, such as for example fibroblast
cells, in a receptacle, prior to the removal of these cells and the
addition of isolated crypts or epithelial stem cells. Examples of
extracellular matrix-producing cells are chondrocytes, producing
mainly collagen and proteoglycans, fibroblast cells, producing
mainly type IV collagen, laminin, interstitial procollagens, and
fibronectin, and colonic myofibroblasts producing mainly collagens
(type I, III, and V), chondroitin sulfate proteoglycan, hyaluronic
acid, fibronectin, and tenascin-C. Alternatively, said ECM is
commercially provided. Commercially provided ECMs are typically
synthetic ECMs. Examples of commercially available extracellular
matrices are extracellular matrix proteins (Invitrogen) and
Matrigel.TM. (BD Biosciences). The use of an ECM for culturing stem
cells enhanced long-term survival of the stem cells and the
continued presence of undifferentiated stem cells.
[0556] An example of an ECM for use in a method of the invention
comprises at least two distinct glycoproteins, such as two
different types of collagen or a collagen and laminin Said ECM can
be a synthetic hydrogel extracellular matrix or a naturally
occurring ECM. A most preferred ECM is provided by Matrigel.TM. (BD
Biosciences), which comprises laminin, entactin, and collagen IV.
Therefore, in some embodiments, the ECM for use in a method of the
invention is a 3D matrix that mimics the extracellular matrix by
its interaction with the cellular membrane proteins such as
integrins.
[0557] Thus in some embodiments, a method of the invention
comprises bringing the stem cell, the population of stem cells or
the isolated tissue fragment and the culture medium into contact
with an extracellular matrix, for example a laminin-containing
extracellular matrix such as Matrigel.TM. (BD Biosciences). In some
embodiments, the culture medium is diffused into the extracellular
matrix.
Compositions and Other Forms of the Invention
[0558] The invention provides a composition comprising a culture
medium according to the invention and stem cells. The invention
also provides a composition comprising a culture medium according
to the invention and organoids. Furthermore, the invention provides
a composition comprising a culture medium according to the
invention and an extracellular matrix.
[0559] The invention also provides a composition comprising a
culture medium of the invention, an extracellular matrix and stem
cells of the invention. The invention also provides a composition
comprising a culture medium of the invention, an extracellular
matrix and one or more organoids of the invention. The invention
also provides a culture medium supplement that can be used to
produce a culture medium as disclosed herein. A `culture medium
supplement` is a mixture of ingredients that cannot itself support
stem cells, but which enables or improves stem cell culture when
combined with other cell culture ingredients. The supplement can
therefore be used to produce a functional cell culture medium of
the invention by combining it with other cell culture ingredients
to produce an appropriate medium formulation. The use of culture
medium supplements is well known in the art.
[0560] The invention provides a culture medium supplement that
comprises an inhibitor according to the invention. The supplement
may contain any inhibitor (or combination of inhibitors) disclosed
herein. The supplement may also contain one or more additional cell
culture ingredients as disclosed herein, e.g. one or more cell
culture ingredients selected from the group consisting of amino
acids, vitamins, inorganic salts, carbon energy sources and
buffers.
[0561] A culture medium or culture medium supplement may be a
concentrated liquid culture medium or supplement (e.g. a 2.times.
to 250.times. concentrated liquid culture medium or supplement) or
may be a dry culture medium or supplement. Both liquid and dry
culture media or supplements are well known in the art. A culture
medium or supplement may be lyophilised.
[0562] A culture medium or supplement of the invention will
typically be sterilized prior to use to prevent contamination, e.g.
by ultraviolet light, heating, irradiation or filtration. A culture
medium or culture medium supplement may be frozen (e.g. at
-20.degree. C. or -80.degree. C.) for storage or transport. In some
embodiments, the culture medium may be stored as a liquid (e.g. at
approximately 4.degree. C.). In some embodiments, the culture
medium may be split and stored as two components: a frozen
component (e.g. at between approximately -20.degree. C. and
approximately-80.degree. C.) and a liquid component (e.g. at
approximately 4.degree. C.). In particular, temperature-sensitive
or time-sensitive degradable material is preferably included in the
frozen component, whereas less sensitive material (for example DMEM
or FCS) can be stored in the liquid form and thus included in the
liquid component for storage and shipping.
[0563] The invention also provides a hermetically-sealed vessel
containing a culture medium or culture medium supplement of the
invention. Hermetically-sealed vessels may be preferred for
transport or storage of the culture media or culture media
supplements disclosed herein, to prevent contamination. The vessel
may be any suitable vessel, such as a flask, a plate, a bottle, a
jar, a vial or a bag.
[0564] The invention also provides a kit comprising a culture
medium, culture medium supplement and/or a composition of the
invention. In some embodiments, the kit further comprises at least
one other additional component, for example selected from the list
comprising: an ECM (for example, Matrigel.TM.), a population of
cells and an organoid.
General
[0565] "GI" numbering is used above. A GI number, or "GenInfo
Identifier", is a series of digits assigned consecutively to each
sequence record processed by NCBI when sequences are added to its
databases. The GI number bears no resemblance to the accession
number of the sequence record. When a sequence is updated (e.g. for
correction, or to add more annotation or information) then it
receives a new GI number. Thus the sequence associated with a given
GI number is never changed.
[0566] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0567] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
[0568] The term "about" in relation to a numerical value x is
optional and means, for example, x+10%.
[0569] Unless specifically stated, a process comprising a step of
mixing two or more components does not require any specific order
of mixing. Thus components can be mixed in any order. Where there
are three components then two components can be combined with each
other, and then the combination may be combined with the third
component, etc.
[0570] Various aspects and embodiments of the invention are
described below in more detail by way of example. It will be
appreciated that modification of detail may be made without
departing from the scope of the invention.
DESCRIPTION OF THE DRAWINGS
[0571] FIG. 1 Mouse colon culture
[0572] a. left: Axing expression is under the control of the Wnt
signaling pathway. Colon crypt organoids of Axin2-LacZ reporter
mice cultured with EGF, Noggin, and R-spondin (ENR) for 3 days.
Absence of LacZ stain indicates that no active Wnt signal is
present in the colon organoids under ENR growth condition. Inset
shows active Wnt signalling visualized by LacZ expression (dark
stain) in freshly isolated colon crypts from the Axin2-LacZ
reporter mice. right: Axin2-LacZ mice derived colon crypts cultured
with ENR+Wnt3A (WENR) for 10 days. Dark stain indicates LacZ
expression in these organoids.
[0573] b. left: Lgr5-GFP-ires-CreER colon crypts cultured with ENR
for 3 days. Absence of GFP fluorescence indicates loss of Lgr5
expression in the colon organoids under ENR growth condition. Inset
shows Lgr5-GFP expression in freshly isolated colon crypts from
Lgr5-GFP-ires-CreER mice. right: Lgr5-GFP-ires-CreER colon crypt
cultured with WENR for 10 days demonstrates the presence of Lgr5
stem cells.
[0574] c. Culture efficiency is determined under three different
conditions: ENR, WENR full crypts, and WENR crypts after mild
enzymatic digestion (WENR digested). Colon crypts were isolated
from proximal colon (black columns) or distal colon (white
columns). *:p<0.05.
[0575] d, e: 4 days after removal of Wnt3A from the WENR culture
medium results in organoid differentiation. d. Chromogranin A (ChA)
in enteroendocrine cells; Mucin2 (muc2) in Goblet cells and the
counter stain with DAPI can be seen. e. Mature enterocytes are
visualized by Alkaline phosphatase staining.
[0576] f. Relative mRNA expression of mature epithelial cell
markers (Vil1 (Villin1), Alpi (Alkalin phosphatase), Chga
(Chromogranin A), Muc2 (Mucin2)) are shown. WENR cultured colon
crypt organoids are cultured for 4 days in WENR (hatched pattern)
or ENR (black) condition. Freshly isolated colon crypts (white) are
used for control. Scale bar in a, b, d, e: 50 .mu.m. Error bars
indicate s.e.m. n=3.
[0577] FIG. 2 Human colon culture
[0578] a. The effect of nicotinamide on human colon crypt
organoids. The majority of human colon crypt organoids die within a
few days in WENR+ gastrin (WENRg) condition (left panel). Addition
of nicotinamide (middle panel: WENRg+nic) improves culture
efficiency and lifespan of human colon organoids. * p<0.001.
nic: nicotinamide.
[0579] b. The effect of small molecule inhibitor for Alk4/5/7
(A83-01) and for the MAP kinase p38 (SB202190) on human colon crypt
organoids. Left panel: Human colon organoids cultured in
WENRg+nicotinamide containing medium form cystic structures 3-4
weeks after culture. Middle panel: Human colon organoids retain
their characteristic budding structure under the Human Intestinal
Stem Cell Culture ("HISC") condition
(WENRg+nicotinamide+A83-01+SB202190). Right panel: A83-01 and
SB202190 synergistically increase number of passages of the human
colon organoids. * p<0.001. N. S.=statistically not significant.
Error bars indicate s.e.m. n=5.
[0580] c. Proliferating cells visualized by the incorporation of
EdU are confined to the budding structures. DAPI is used as a
counterstain
[0581] d. Representative picture of a karyotype from a 3-month-old
human colon crypt organoid. Scale: 100 .mu.m.
[0582] e. Heat-map of the expression profile of cultured human
intestinal organoids. The heat-map is a comparison of human small
intestinal crypts and human small intestinal villi. Genes more
highly expressed in the crypt are dark grey (top-half of heat-map),
genes more highly expressed in the villus are light grey
(bottom-half of the heat-map). Organoids cultured in-vitro clearly
exhibit a similar expression profile to freshly isolated small
intestinal crypts and express known stem cell markers. (lane 1:
human small intestinal organoids #1, lane 2: human small intestinal
organoids #2, lane 3: human colon organoids, lane 4: freshly
isolated human small intestinal crypts. The four samples are
compared to human smallintestinal villus).
[0583] FIG. 3 Human intestinal organoid cell type composition
[0584] (a-c) Human organoids differentiate into the different cell
types of the intestine after withdrawal of Nicotinamide and
SB202190. Markers of the different cell types were used to
demonstrate differentiation. (a) Top panel: Alkaline phosphatase
staining for mature enterocytes, Middle panel: PAS staining for
goblet cells, Bottom panel: Synaptophysin staining for
enteroendocrine cells. (b) In each case, the light areas indicate
staining. Mucin2 (Muc2) staining in the middle panel represents
goblet cells and Chromogranin A (ChgA) in the left-hand panel
represents enteroendocrine cells (see arrow and inset). DAPI is
used as a counterstain (right panel). (c) Lysozyme (Lysz) is
stained in the left-hand panel to show Paneth cells. DAPI is used
as a counterstain (right panel).
[0585] (d-f) Goblet cell differentiation (Muc2) is blocked by
SB202190 treatment of organoids (d), while the Notch inhibitor DBZ
increases goblet cell number in the human organoids (1).
Proliferating cells are represented by EdU incorporation (middle
panel) are increased in SB202190 treated organoids (d) or decreased
in DBZ treated organoids (0. Organoids are cultured under the
following conditions for 5 days: a) top:
ENRg+A83-01+SB202190+Nicotinamide, a) middle and bottom, b), c)
WENRg+A83-01, d)WENRg+A83-01+SB202190, e) WENRg+A83-01, f)
WENRg+A83-01+DBZ. Scale bar:20 .mu.m (a), 50 .mu.m (b-f). a, b,
d-f: human colon crypt organoids, c: human small intestinal
organoids.
[0586] FIG. 4 Adeno(carcino)ma cultures
[0587] a. Lgr5-GFP-Tres-CreER/APCfl/fl crypts cultured with EGF (E)
(top) or EGF+Noggin (EN) (bottom) for 10 days. b. Relative mRNA
expression of Lgr5 and Axin2. Freshly isolated adenoma cells
(white) were cultured with EGF (hatched) or EGF+Noggin (black). c.
Culture efficiency of organoids from sorted Lgr5-GFPhi, Lgr5-GFPlo,
Lgr5-GFP-ve cells. *p<0.01. one way ANOVA. Error bars indicate
s.e.m. n=3
[0588] d. Time course culture of human colon adenocarcinoma
cells.
[0589] FIG. 5 Culture of Barrett's esophagus and treatment with
Notch inhibitor.
[0590] a. Isolated epithelium from Barrett's esophagus (BE)
cultured with HISC condition for 7 days forms cystic structures. b.
Addition of FGF10 significantly increases the number of passages
for BE organoids. Error bars indicate s.e.m. n=3 c. Representative
time course of a BE organoid. d. Paraffin sections from BE
organoids. Nicotinamide and SB202190 are withdrawn for 4 days with
(right) or without (left) the Notch inhibitor DBZ added to the
medium. Proliferating cells (Ki67 stain) disappear and PAS+ goblet
cells increase with DBZ treatment.
[0591] FIG. 6 Axin2 mRNA expression is recovered in mouse colon
organoids under the presence of Wnt-3A
[0592] Isolated colonic crypts are analysed for Axin2 mRNA
expression after 3 days or 7 days culture with ENR (hatched) or
WENR (black). Freshly isolated colon crypts were used as control.
Error bars indicate s.e.m. n=3
[0593] FIG. 7 Relative mRNA expression of mature epithelial cell
markers
[0594] Freshly isolated small intestinal crypts (white) are
cultured in HISC condition for 14 days, followed by a culture with
the indicated culture condition for 4 days. mRNA expression of ALPI
(Alkaline phosphatase), VIL1 (Villin 1), LYZ (Lysozyme), CHGB
(Chromogranin B) and MUC2 (Mucin2) was analysed. Culture condition:
HISC (black), ENRg+A83-01+SB202190+Nicotinamide, WENRg+A83-01,
ENRg+A83-01, ENRg. Freshly isolated small intestinal crypts were
used as control (set as 1.0 for ALPI, VIL1 and LYZ, as 5.0 for CHGB
and MUC2. Error bars indicate s.e.m. n=3.
[0595] FIG. 8 Sorted Lgr5-GFP-cells form Lgr5-GFP+organoids
[0596] Single sorted Lgr5-GFP-APCfl/fl adenoma cells are cultured
with EGF+Noggin (EN) or EGF (E) for 7 days. Adenoma organoids
derived from Lgr5-GFP-cells recovered Lgr5-GFP expression under EN
condition but not under E condition (a, c: bright, b, d: GFP
autofluorescence).
[0597] FIG. 9 Histochemical analysis of adenoma/colon cancer
organoids
[0598] Mouse small intestinal adenoma organoids (Left panel) and
human colon cancer organoids (Right panel) were analyzed with
indicated histochemical (HE, PAS and Alkaline phosphatase) or
immunohistochemical (Chromogaranin A, Ki67 and Caspase3)
stainings.
[0599] FIG. 10 Paneth cells in BE organoids
[0600] Lysozyme+Paneth cells were observed in differentiated BE
organoids.
[0601] FIG. 11 List of reagents used for organoid culture
[0602] FIG. 12 List of reagents used for optimization of human
intestinal organoid culture
[0603] FIG. 13 List of small molecule inhibitors used for
optimization of human intestinal organoids culture
[0604] FIG. 14 List of the 25 most up- and down-regulated genes
[0605] mRNA from human small intestinal organoids or colon
organoids are compared with that from freshly isolated small
intestinal villi by microarray. The 25 most upregulated and
downregulated genes are shown. Hatched lines highlight genes which
were in the top 70 most upregulated and downregulated genes in
freshly isolated human small intestinal crypts vs. villi.
[0606] FIG. 15. Summary of proliferation, differentiation and
apoptosis status of each organoid culture condition
[0607] FIG. 16: Microarray comparison of mouse pancreatic
organoids
[0608] A--Microarray clustering analysis, comparing RNA from the
pancreas organoids (cultured in the conditions described in Example
2) with adult pancreas, adult liver and newborn liver. From left to
right: i) pancreas organoid; ii) adult pancreas; adult liver
(sample 1 [S1] and sample 2 [2]); iv) adult liver S2; and v)
newborn liver.
[0609] B--Raw signal data from the microarray analysis, comparing
the expression levels of selected ductal markers, transcription
factors necessary for Ngn3 expression and endocrine markers in
adult liver, adult pancreas, pancreas organoids and liver organoids
in expansion media.
[0610] FIG. 17: The effect of Noggin on the expansion of pancreatic
organoids
[0611] A--Bar charts showing gene expression analysis of pancreatic
organoids cultured in EGFRA so, that have never been cultured with
Noggin (black) with organoids cultured in EGFRAN, so have always
been cultured with Noggin (white). The effect of culturing the
pancreatic organoids in EGFRA for 2 days and then withdrawing
Noggin and culturing for a further 2 or 4 days (light grey) and the
effect of culturing the pancreatic organoids in EGFRA for 2 days
and then adding Noggin and culturing for a further 2 or 4 days
(dark grey) on gene expression is also shown. mRNA levels
(arbitrary units) are presented on the Y axis. mRNA of the
following early endocrine markers is analysed in the main figure:
Sox9, Hnf1b, Hnf6, Hnf1a, Nkx2.2, Nkx6.1 and Pdx1. mRNA of the
following ductal markers in analysed in the inset part: keratin 7
(Krt7) and keratin 19 (Krt19).
[0612] B--Bar chart showing the effect of Noggin on the expression
of Lgr5 in pancreatic organoids in the expansion culture medium.
Data are provided for pancreatic organoids cultured in EGFRA that
have never been cultured with Noggin (black) with organoids
cultured in EGFRAN and so have always been cultured with Noggin
(white). The effect of culturing the pancreatic organoids in EGFRAN
and then withdrawing Noggin and culturing for a further 6 days
(light grey) and the effect of culturing the pancreatic organoids
in EGFRA and then adding Noggin and culturing for a further 6 days
(dark grey) on Lgr5 gene expression is also shown. mRNA levels
(arbitrary units) are presented on the Y axis.
[0613] FIG. 18: Human insulin producing cells develop from ex vivo
expanded, in vivo transplanted progenitor cells
[0614] A--Growth of human pancreas tissue from progenitor cells
(pancreas stem cells) at P0: (Day 1); P0: (Day 5); P1: (Day 12) and
P3: (Day 24), where "P" refers to the number of passages.
[0615] FIGS. 18B and C show transplantation of human pancreatic
organoids under the murine peri-renal capsule.
[0616] B--3 hours after transplantation of the pancreatic organoid
cells into the recipient mice: DAPI (nuclear marker) staining in
the upper picture indicates all cells; K19 (ductal marker) staining
in the lower picture shows all transplanted cells and insulin (beta
cell marker) in the lower picture indicates insulin-producing
cells.
[0617] C--1 month after transplantation of the pancreatic organoid
cells into the recipient mice: DAPI (nuclear marker) staining in
the upper picture (in blue) indicates all cells; CK19 (ductal
marker) expression in the middle picture (in green) indicates all
transplanted cells and insulin (beta cell marker) in the lower
picture (in red) indicates insulin-producing cells. A selection of
the insulin-producing cells are encircled but all clearly stained
cells are thought to be insulin positive.
[0618] FIG. 19: Pancreatic organoid gene expression
[0619] This table shows the pancreatic gene expression of the most
upregulated genes when compared to liver organoids.
[0620] FIG. 20: Mouse liver organoid culture shows stable
karyotyping after long-term culture.
[0621] A--DIC images of liver organoids maintained in EGF (E) and
R-spondin 1 (R), supplemented with FGF10, HGF and Nicotinamide
(left figure, ER) or maintained in the same combination
supplemented with Noggin (N) and Wnt3Aconditioned media (W) (right
figure, ENRW) for a period of 24 months.
[0622] B--Karyotype analysis of mouse liver organoids after 8
months in culture. Normal chromosomal counts (n=40, left panel
figure) and polyploidy, a typical hepatocyte feature, were found
(n=80, right panel figure)
[0623] FIG. 21: Supplemental factors FGF10, HGF and Nicotinamide;
effect on liver organoid growth and differentiation.
[0624] A--Diagram depicting the genes differentially expressed
during the 3 stages of liver development, from hepatoblast to
mature hepatocyte.
[0625] B--Scheme showing the protocol used. Cultures were seeded in
expansion medium (ERFHNic: EGF (E) and R-spondin 1 (R),
supplemented with FGF10, HGF and Nicotinamide; ERFHNic is indicated
as `ER` in FIG. 8B) 2 days prior the experiment. Two days later,
culture media was changed to either EGF (E) alone or EGF
supplemented with R-spondin 1 (ER) with or without additional
supplements chosen from FGF10 (F) or HGF (H) or Nicotinamide (Nic)
or a combination of these at the concentrations stated in the text.
Five days later cultures were split and replated at 1:4 ratio for
each condition. Under these conditions, cultures have been split
and replated every 7 days for a total period of 10 weeks
[0626] C--First day after first split in each of the culture
conditions tested. Results shows that EGF and Rspondin 1 combined
with FGF10 or HGF or Nicotinamide or a combination of these are
essential to achieve at least 1 passage.
[0627] D--After long-term culture, the combination of ER
supplemented with FNic or ER supplemented with FHNic, both result
in high passage numbers. After passage 10, the growth rate is
better for the culture condition including the 3 supplemental
factors; ERFHNic.
[0628] E--RT-PCR analysis showing the expression of different
hepatocyte markers (CYP3A11, A1b, FAH) and cholangiocyte marker
(K19) 5 days after the withdrawal of certain factors (starting
point was ERFHNic). Note that only the condition EF showed
expression of all hepatocyte markers tested. HPRT was used as a
housekeeping gene to normalize for gene expression.
[0629] FIG. 22: Table showing the quantification of different
hepatocyte and cholangiocyte specific transcription factors in
cells from three different liver culture conditions and in adult
liver tissue. Also shown is the expression of the key components of
the Notch and TGF-beta signalling pathways. E=EFHNic, ER=ERFHNic,
ENRW=ENRWFHNic.
[0630] FIG. 23: Differentiation protocol
[0631] A--Scheme showing the protocol used. Cultures were seeded in
expansion medium (ERFHNic: EGF (E) and R-spondin 1 (R),
supplemented with FGF10, HGF and Nicotinamide; ERFHNic is indicated
as `ER` in FIG. 10A) 2 days prior to the experiment. Two days
later, culture media was changed to EGF (E) supplemented with
either A8301 (A), or DAPT (D), or FGF10 (F) or HGF (H) or
Nicotinamide (Nic) or R-spondin 1 (R) or Wnt3A or Noggin (N) or a
combination of these at the concentrations shown. RNA was isolated
at several time points. Mouse liver tissue was taken as positive
control (+) whereas water was taken as negative control (-).
[0632] B--RT-PCR analysis showing the expression of the hepatocyte
markers CYP3A11, A1b, FAH, tbx3, TAT and Gck 7 days after
differentiation conditions. Note that only the condition EADF
showed an expression of all hepatocyte markers tested. HPRT was
used as a housekeeping gene to normalize for gene expression.
[0633] C--Time course expression analysis after differentiation
conditions. At days 2, 5 and 8 days after differentiation, the
expression of the hepatocyte markers CYP3A11, Alb, FAH, and the
cholangyocyte marker K19, was analysed by RTPCR. Note that the
expression of the liver markers CYP3A11 and FAH starts at day 5 and
peaks at day 8 after. HPRT was used as a housekeeping gene to
normalize for gene expression. A; A8301, D; DAPT, F; FGF10, H; HGF,
De; Dexamethasone
[0634] D--Titration experiment showing the expression of the
hepatocyte markers CYP3A11, Alb, FAH, tbx3, TAT, G6P and Gck 7 days
after different concentrations of the differentiation compounds A
and D. HPRT was used as a housekeeping gene to normalize for gene
expression.
[0635] E--Immunofluorescent staining for the liver markers K19,
Albumin and hepatocyte surface marker
[0636] F--Xgal staining on Albcreert2LacZ mice liver-derived
organoids. Albumin positive cells (arrows) were detected after EADF
differentiation in tamoxifen induced Albcreert2LacZ derived
cultures.
[0637] FIG. 24: Prostaglandin signalling pathway (Antagonism of the
prostaglandin D.sub.2 receptors DP.sub.1 and CRTH2 as an approach
to treat allergic diseases. Roy Pettipher, Trevor T. Hansel &
Richard Armer Nature Reviews Drug Discovery 6, 313-325 (April
2007)).
[0638] FIG. 25: Liver organoids cultured in (A) basal medium
comprising hEGF (100 ng/ml, Invitrogen); human noggin (hnoggin) (25
ng/ml, peprotech); gastrin (10 nM, sigma); hFGF10 (peprotech);
nicotinamide (10 mM, sigma); A8301 (500 nM, Tocris); hHGF (50
ng/ml, peprotech); Rspo conditioned media (10%); (B) basal
medium+PGE2 (50 nM); (C) basal medium+CHIR99021 (3 uM); (D) basal
medium+CHIR99021 (3 uM)+PGE2 (50 nM).
[0639] FIG. 26: Liver organoids cultured in basal medium (as
described for FIG. 25) with and without Arachidonic acid.
[0640] FIG. 27: Gene expression profile of mouse liver organoids
under differentiation conditions resemble the adult and newborn
liver profile
[0641] A--Gene clusters showing the genes similarly expressed (a)
or similarly shut down (b) between the differentiation condition
EADF and adult or newborn liver.
[0642] B--Gene clusters showing the genes differentially expressed
between the liver organoids and adult or newborn liver (a) and the
genes similarly expressed between EADF and newborn liver (b).
[0643] C--Raw signal data from a microarray analysis, comparing the
expression levels of selected ductal markers, transcription factors
necessary for Ngn3 expression and endocrine markers in adult liver,
adult pancreas, pancreas organoids and liver organoids in expansion
media.
[0644] FIG. 28: Mouse liver signature genes
[0645] Table showing a) markers expressed in mouse liver stem
cells; b) markers not expressed in mouse liver stem cells; c)
hepatocyte and cholangiocyte markers expressed in mouse liver stem
cell signature for mouse liver organoids in expansion media; d)
hepatocyte and cholangiocyte markers not expressed in mouse liver
stem cell signature for mouse liver organoids in expansion media;
e) reprogramming genes expressed in mouse liver organoids; f)
reprogramming genes not expressed in mouse liver organoids. The
results were obtained using a liver microarray using the Universal
Mouse Reference RNA (Strategene, Catalog #740100) as a reference
RNA. If the absolute figures detected were less than 100, the gene
was consider as undetected.
[0646] FIG. 29: Human liver signature genes
[0647] Table showing results of liver mircroarray of human
organoids. From left to right, the results are shown for a)
expansion medium EM1, b) expansion medium EM2, c) differentiation
medium, d) adult liver.
The numbers (log 2) in the left four columns are the result of a
comparison between the sample and a reference (commercial) RNA
sample which is used for all arrays. The relative expression of
mRNA in each sample compared to the RNA present in the reference
sample is shown. The reference RNA used was Universal Human
Reference RNA (Stratagene, Catalog #740000). Thus, negative numbers
in these columns do not relate to real expression levels it just
means there is less of that RNA then in the Reference sample. The 4
columns on the right are absolute figures. If they are below 100,
they are considered as undetected.
[0648] FIG. 30: Morphology of liver organoids. (A) Upper panels:
paraffin section of a mouse liver showing the different domains
(PT=portal triad, CV=central vein). Lower panels: Paraffin section
of a liver organoid showing different domains b (single layered
epithelia) and h (stratified epithelia) (B) Right pannel: Ecadherin
staining in the liver organoids. Two different domains can be
identified. Domain b, formed by a single layered epithelia that
resembles the bile duct structures in the liver. This bile duct
domain is formed by highly polarized cells that shows positive
staining for pancytokeratin (PCK) (lower panel). Left panels show
the presence of a second domain within the liver organoids. This h
domain is formed by a stratified epithelia with non-polarized
cells. The cells are organized around a central lumen and express
the hepatocyte marker Alb. Magnification 10.times..
[0649] FIG. 31: H&E staining of pancreas organoids
[0650] Mouse pancreas organoids were cultured in expansion
conditions EGFNRA83-01 [(EGF(50 ng/ml), Gastrin (50 nM), Noggin
(10%), Rspondin (5%), FGF10 (100 ng/ml) and A8301 (50 nM)] during 8
passages (.about.10 weeks). The organoids were removed from the
matrigel using
[0651] BD Cell Recovery Solution following manufacturer's
instructions and fixed with 4% paraformaldehyde at room temperature
during 1 h. Then, the organoids were washed three times with cold
PBS, dehydrated with increased concentration of alcohol and
embedded in paraffin. 3 um parafine sections were stained with
Hematoxyline-Eosine to analyze the histology of the pancreas
organoids.
[0652] A strong variability in the shape and structure of the
organoids was observed. Some of the organoids are cystic structures
formed by a monolayer of polarized epithelial cells. Other
organoids show the same monolayer of epithelial cells and some
stratified areas where cells are smaller in size and with a round
shape. In some organoids invaginations occupying the inner space of
the cystic structure were observed.
[0653] The stainings show that some organoids comprise mostly
monolayers of epithelial cells (left bottom), whereas other
organoids comprise stratified regions and/or pseudo stratified
regions and/or folded monolayers (right bottom). Most pancreatic
organoids comprise regions of stratified cells and monolayers
(sometimes folded).
[0654] FIG. 32. Quantification of forskolin-induced murine small
intestine organoid swelling. (a)
[0655] Light microscopy analysis of organoids stimulated with
forskolin or DMSO. Representative examples for the indicated time
points after start of stimulation are shown. The red line indicates
the internal organoid lumen. (b) Fluorescence confocal image of a
calcein-green-labeled organoid with object recognition (green line)
by image analysis software. (c) Representative example of a
forskolin-stimulated calcein-green-labeled organoid. Differential
interference contrast (DIC) and fluorescence was imaged using live
cell confocal microscopy. Surface area relative to t=0 is indicated
in the top-left corner. (d) The surface area relative to t=0
(normalized area) of 11 individual organoids in a single well. The
average is indicated in black (mean.+-.s.e.m.). (e) Dose-dependent
increase of surface area by forskolin (5 .mu.M (n=4 number of
organoids analyzed), 5.times.10.sup.-2 .mu.M (n=11),
5.times.10.sup.-4 .mu.M (n=10), DMSO n=9)). Scale bars (a-c) 30
.mu.m. All results are representative for at least three
independent experiments.
[0656] FIG. 33. Forskolin-induced swelling of murine organoids is
CFTR dependent. (a)
[0657] Normalized swelling curves of forskolin-stimulated
calcein-green-labeled organoids pre-incubated with DMSO (n=8),
CFTR-.sub.inh172 (n=7), GlyH-101 (n=9) or both CFTR-.sub.inh172 and
GlyH-101 (n=11) (mean.+-.s.e.m.). (b) Representative confocal
microscopy images of calcein-green labeled wild type or
CFTR-deficient organoids in response to forskolin. Scale bars 50
.mu.m.
[0658] (c) Quantification of forskolin-induced swelling in wild
type (n=6) or CFTR-deficient (n=11) organoids (mean.+-.s.e.m.)
(d,e) Similar to b,c but for wild type (n=8) and CFTR-F508del
(n=12) organoids. Scale bars 30 .mu.m. (f) Absolute size of wild
type or CFTR-deficient organoids quantified in (c) at t=0
(mean.+-.s.e.m.). (g) Forskolin-stimulated swelling of
calcein-green labeled CFTR-F508del organoids cultured at 37.degree.
C. with (n=20) or without (n=15) CFTR inhibition or cultured at
27.degree. C. for 24 hours with (n=31) or without (n=27) CFTR
inhibition (mean s.e.m.). (h) Forskolin-induced swelling of
calcein-green labeled CFTR-F508del organoids pre-incubated for 24
hours with DMSO with (n=15) or without (n=18) CFTR inhibition or
pre-incubated with the CFTR corrector compound VRT-325 with (n=14)
or without (n=26) CFTR inhibition (mean.+-.s.e.m.). (i) Normalized
forskolin-induced swelling of CFTR-F508del organoids pre-treated
for 24 hours with DMSO (n=16), VRT-325 (n=18), Corr-4a (n=20) or
both correctors (n=20) (mean+s.e.m.). All results are
representative for at least three independent experiments.
[0659] FIG. 34. Forskolin-induced swelling in human healthy control
or cystic fibrosis organoids. (a) Quantification of
forskolin-induced organoid swelling pre-incubated with DMSO,
CFTR.sub.inh-172, GlyH-101 or both CFTR.sub.inh-172 and GlyH-101
(n=5, n=7, n=8, n=10) (mean s.e.m.). (b) Forskolin-stimulated
swelling of organoids derived from 4 individual healthy controls
(1-4: n=30, n=18, n=13, n=42) and 1 CF CFTR-F508del homozygous
patient (n=30) (mean+s.e.m.). (c) Normalized swelling of
forskolin-induced calcein-green labeled CFTR-F508del organoids
cultured for 24 at 37.degree. C., or at 27.degree. C. with or
without CFTR inhibition (n=10 for all conditions) (mean.+-.s.e.m.).
(d) Representative confocal microscopy images of calcein-green
labeled HC-derived or CF patient-derived organoids in response to
forskolin upon pharmalogical manipulation of CFTR. Scale bars 60
.mu.m. (e) Normalized forskolin-induced swelling of CFTR-F508del
organoids pre-treated for 24 hours with DMSO, VRT-325, Corr-4a, or
both correctors (n=10 for all conditions) (mean+s.e.m.). (f) CF
patient-derived organoid swelling in response to forskolin with or
without 24 hour pre-treatment of corrector VX-809, VX-770
stimulation (simultaneous with forskolin) or combined VX-809 and
VX-770 treatment (n=10 for all conditions) (mean.+-.s.e.m.). (g)
Forskolin-induced swelling of organoids upon DMSO treatment
(control) or combined compound treatment from e and f, compared to
HC organoids (n=10 for all conditions) (mean+s.e.m.). Each line in
(d) represents organoid swelling averaged form at least three
independent experiments per individual. Results from all other
figures are representative for at least three independent
experiments.
[0660] FIG. 35. Light microscopy analysis of wild type murine
organoids stimulated with forskolin or DMSO. Representative
examples for the indicated timepoints after start of stimulation
are shown. The forskolin-induced swelling (FIS) of organoids was
reversed upon removal of forskolin by washing.
[0661] FIG. 36. CFTR mRNA is expressed in mouse and human
organoids. The bars show real-time PCR cycle threshold (CT) values
representing mRNA levels of CFTR, .beta.2m or GAPDH isolated from
CFTR-F508del (left graph) or CFTR-/-(middle graph) organoids and
their corresponding wild types, or human organoids.
[0662] FIG. 37. Gradual forskolin-induced swelling prevents
organoid collapse. Normalized surface area increase of individual
forskolin-stimulated (a) wild type, (b) CFTR-F508del
(temperature-rescued) and (c) human (5% Wnt3a-conditioned medium,
WCM) organoids. The averaged forskolin-induced swelling of
different organoid types was analyzed up to 10 (FIG. 1d,e+2a,c,e),
20 (FIG. 2g) or 40 (FIG. 2h,i+2a-c) minutes (dashed line).
[0663] FIG. 38. Increased FIS by treatment of corrector compounds
is CFTR dependent. Forskolin-induced swelling of calcein-green
labeled human CFTR-F508del organoids pre-incubated for 24 hours
with DMSO, or with both VRT-325 and Corr-4a with or without CFTR
inhibition (n=10 for all conditions) (mean.+-.s.e.m.). Results are
representative for at least three different experiments.
[0664] FIG. 39. Cholera toxin-induced organoid swelling in human
organoids is CFTR dependent. Forskolin and cholera toxin induce
swelling of HC-derived organoids, but not of CFTR-F508del
organoids. The cholera toxin response is delayed compared to
forskolin because of its apical extracellular function. (n=10 for
all conditions) (mean.+-.s.e.m.). Results are representative for at
least three different experiments.
[0665] FIG. 40. Human organoids in normal or reduced Wnt3a culture
conditions. (a) Light microscopy images of human organoids cultured
at normal (50%, left panel) or reduced (5%, right panel) Wnt3a
conditioned medium (WCM) concentrations. Scale bars 400 .mu.m. (b)
Representative examples of forskolin-induced swelling at normal or
reduced Wnt3a conditions. Scale bars 50 .mu.m. The dashed line
depicts the internal organoid lumen (c) Quantification of
forskolin-induced organoid swelling at normal Wnt3a levels
pre-incubated with DMSO, CFTR.sub.inh-172, GlyH-101 or both
CFTR.sub.inh-172 and GlyH-101 (n=29, n=41, n=26, n=15)
(mean.+-.s.e.m.). (d) Quantification of forskolin-induced swelling
of low-passage budding organoids cultured at 50% (n=9) or 5% (n=12)
Wnt3a conditioned medium (WCM) concentrations averaged from two
independent experiments (mean.+-.s.e.m.). All results are
representative for at least three independent experiments.
[0666] FIG. 41. H&E stains of prostate organoids
[0667] Tissue fragments of mouse prostate epithelium were embedded
in matrigel. The expanding cells were split weekly. The culture can
be maintained for extended period of time without loosing genetic
stability or proliferation capacity. FIG. 41 shows a comparison of
the prostate epithelium of the prostate itself and the organoid
cultures after periods of three months. The mouse prostate
organoids grow in media containing ENR (EGF, Noggin and Rspondin)
in the presence or absence of testosterone. Human culture requires
the addition of a TGF-beta inhibitor. The H&E of fixation and
embedding in paraffin demonstrates the different levels of
stratification and folding of the epithelium in vivo. The cultured
prostate organoids show similar diversity in folding and
stratification.
[0668] FIG. 42. CK8 (a differentiation marker) stains of prostate
organoids
[0669] Tissue fragments of mouse prostate epithelium were embedded
in matrigel. The expanding cells expand were split weekly. The
culture can be maintained for extended period of time without
loosing genetic stability or proliferation capacity. FIG. 42 shows
the presence of CK8 expressing luminal cells. The cultures are
grown in media containing ENR (EGF, Noggin and Rspondin) in the
presence or absence of testosterone. Human culture requires the
addition of a TGF-beta inhibitor. The addition of testosterone
allows for the differentiation into CK8 positive luminal cells
while at the same time stimulating stem cell maintenance and
expansion. Testosterone also increases the stratification and
folding of the epithelium.
[0670] FIG. 43. Mouse prostate after 25 weeks in culture.
[0671] Prostate organoids grown in the EGF, Noggin, Rspondin, NAC,
B27, Glutamin/max, pen/strep, Ad-DMEM/F12+testosterone. The shape
of the organoid is determined by origin of tissue (position in the
prostate before isolation). The prostate consists of different
lobes or regions. The different regions display specific epithelial
structures (stratification and folding), After in vitro culturing
the organoids appear to maintain the different macroscopic
structure (stratified or folded) of the part of the prostate from
which it originated.
[0672] FIG. 44. PCR of 3 week human prostate culture
[0673] Normal and cancerous prostatic epithelium was isolated and
grown for three weeks in ENR FGF10, ENRF+DHT, WENRF, WENRF+DHT,
ENR, ENR+DHT culture conditions. All culture conditions contained
A83, P38i and Nicotinamide.
[0674] FIG. 44(a): RNA was isolated and RT-PCR was performed for
markers of prostatic epithelium. In both normal and tumor tissue
the luminal markers CK18, CK8 and B-MSP are expressed. All culture
conditions express the AR. In normal tissue addition of DHT
increases AR expression in all culture conditions. In tumor tissue
AR expression is not influenced by DHT addition. In all culture
conditions basal epithelial markers CK14, CK5 and p63 are
expressed. Putative stem cell marker LGR5 is expressed under ENRF
conditions in normal tissue. In tumor tissue LGR5 expression is
induced with the addition of DHT in all culture conditions.
TNFRSF19, also a putative stem cell marker, is expressed in all
conditions in normal and tumor tissue. The prostate specific
transcription factor NKX3.1 is expressed in all conditions.
Addition of testosterone increases growth/doublings while
maintaining markers for the different cell type of the prostate
(basal and luminal)
[0675] FIG. 44(b): Two representative pictures of human organoids
grown under ENRF+1 nM DHT conditions [0676] Lane 1: Line 1 ENRF
Normal tissue [0677] Lane 2: Line 1 ENRF+1 nM DHT Normal tissue
[0678] Lane 3: Line 2 WENRF Normal tissue [0679] Lane 4: Line 2
WENRF+1 nM DHT Normal tissue [0680] Lane 5: Line 3 ENR Normal
tissue [0681] Lane 6: Line 3 ENR+1 nM DHT Normal tissue [0682] Lane
7: Whole prostate Normal tissue [0683] Lane 8: H.sub.2O [0684] Lane
9: Line 1 ENRF Tumor tissue [0685] Lane 10: Line 1 ENRF+1 nM DHT
Tumor tissue [0686] Lane 11: Line 2 WENRF Tumor tissue [0687] Lane
12: Line 2 WENRF+1 nM DHT Tumor tissue [0688] Lane 13: Line 3 ENR
Tumor tissue [0689] Lane 14: Line 3 ENR+1 nM DHT Tumor tissue
[0690] Lane 15: Whole prostate Tumor tissue [0691] Lane 16:
H.sub.2O
[0692] FIG. 45. PCR of mouse prostate organoid
[0693] Three biologically independent lines were cultured under ENR
or ENR+1 nM DHT conditions. RNA was isolated and RT-PCR was
performed for markers of prostatic epithelium. In both culture
conditions luminal prostate markers Cytokeratin 18 (CK18) and
Cytokeratin 8 (CK8) are broadly expressed. Androgen Receptor (AR)
is expressed in both conditions. Basal markers p63 and Cytokeratin
5 (CK5) are expressed in both culture conditions, but upon addition
of DHT basal markers are downregulated. Putative stem cell markers
Lgr5 and Tnfrsf19 are downregulated upon addition DHT. However
under these conditions sternness is maintained while differentiated
cells are also present. These conditions allow unlimited cell
expansion (for now 9 months at population doublings 2,5 a week).
All cultures are positive for the prostate specific marker Nkx3.1.
Addition of testosterone increase growth/doublings up to 3 fold
while maintain markers for the different cell type of the prostate
(basal and luminal) [0694] Lane 1: Line 1 ENR [0695] Lane 2: Line 1
ENR+1 nM DHT [0696] Lane 3: Line 2 ENR [0697] Lane 4: Line 2 ENR+1
nM DHT [0698] Lane 5: Line 3 ENR [0699] Lane 6: Line 3 ENR+1 nM DHT
[0700] Lane 7: Whole mouse prostate [0701] Lane 8: H.sub.2O
[0702] FIG. 46: Stomach (gastric) organoids
[0703] Human stomach organoids. Tissue was isolated from the
corpus. The cells were culture in the stomach organoid medium (EGF,
Noggin, Rspondin, Wnt, Nicotinamide, FGF10, Gastrin, TGF-beta
inhibitor (A8301). Cells are split weekly.
[0704] FIG. 46(a): This picture was taken after 2 months of
culturing.
[0705] FIG. 46(b): H&E and different antibody staining after
fixation and paraffin sectioning of a culture after 2 weeks of
culturing. It shows the presence of the following cells: PAS for
Mucin producing cells; Muc5Ac for Surface mucous pit cells; Much
for mucous neck cells. The H&E stain shows a single layer of
polarized epithelium.
[0706] FIG. 47: Isolation of prostatic tissue See example 8 (Photos
from UCSF)
[0707] FIG. 48: A) Mouse organoids were cultured in the presence of
different doses recombinant mouse RANKL for 72 h. mRNA expression
levels for RANK, the transciption factor SpiB and the M
cell-specific markers GP2 and AnnexinV were determined by qPCR. B)
Confocal analysis of GP2 (see arrow) and AnnexinV (see arrow)
expression in mouse organoids cultured with 100 ng/ml RANKL for 72
h.
[0708] FIG. 49: Human organoids were cultured in the presence of
different doses of recombinant human RANKL for 7 days. mRNA
expression levels for RANK, SipB and the M cell-specific marker GP2
were determined by qPCR. EM: Expansion medium; DM: differentiation
medium.
EXAMPLE 1
[0709] To address the need for improved culture media and methods
for human epithelial stem cells, the inventors investigated
signalling pathways that are known to be subverted in certain
cancers e.g. colorectal cancer. It was hypothesised that these
pathways, which affect cell fate in cancer, may also play a role in
determining cell fate under in vitro cell culture conditions.
[0710] In a first screening experiment, a series of vitamins,
hormones and growth factors were tested in combination with
standard stem cell culture media. Gastrin and nicotinamide were
identified as resulting in significantly improved culture
conditions. Incorporating these factors into the standard culture
conditions, a second screening experiment was performed, in which
certain small molecule inhibitors related to relevant signalling
pathways, such as ERK, p38, INK, PTEN, ROCK, and Hedgehog, were
tested. In the present state of the art, there would be no
reasonable way to predict what the outcome of each of these
additional compounds would be on the culture medium properties.
TABLE-US-00002 TABLE 2 List of reagents used for optimization of
human intestinal organoids culture Description Source Concentration
Activity* First screening (WENR**) Hormones, vitamins etc
Hydrocortison Sigma 500 nM 0 Gastrin*** Sigma 1 uM 1+ Exendin4 GLP1
analog Sigma 100 nM 0 Nicotinamide Vitamin Sigma 10 mM 3+ B
derivative L-Ascorbic Vitamin C Sigma 10 uM 0 acid anti-oxidant
Sigma 1x 0 mixture Lipid mixture Sigma 1x 0 PGE2 Sigma 10 uM 1+
(Cystic) Cholera Toxin Sigma 100 nM 1+ (Cystic) Growth factors BDNF
Peprotech 100 ng/ml 0 GDNF Peprotech 100 ng/ml 0 FGF2 Peprotech 100
ng/ml 0 FGF10 Peprotech 100 ng/ml 0 Follistatin Peprotech 100 ng/ml
0 Cyr61 Peprotech 1 ug/ml 0 LIF Millipore 1000 U/ml 0 Second
screening (WENR + gastrin + Nicotinamide) Small molecule inhibitors
PD98059 ERK inhibitor Sigma 10 uM 1- SB203580 p38 inhibitor Sigma
1-10 uM 2+ SB202190 p38 inhibitor Sigma 1-10 uM 2+ SP600125 JNK
inhibitor Sigma 10 uM 0 PS48 PDK1 activator Sigma 5 uM 0 Y27632
ROCK Sigma 10 uM 1+ inihibitor cystic Cyclopamine Hedgehog Sigma
100 nM 1- inhibitor 5 Azacytidin DNA Stemolecule 1- methylase
inhibitor Dorsomorphin BMP inhibitor Stemolecule 0 A83-01 ALK4, 5,
7 Tocris 50n-1 uM 3+ inhibitor VO-OHpic PTEN inhibitor Sigma 500 nM
3- trihydrate Pifithrin-.alpha. p53 inhibitor Sigma 0 BIX01294 G9a
HMTase Stemolecule 1- inhibitor *Activity scale (plating efficiency
was compared with control after 4 days culture): 0 = no change; 1+
= <50% increase; 2+ = 50-100% increase; 3+ = >100% increase;
1- = 0-50%; 2- = 50-100% decrease; 3- = >100% decrease. **WENR
comprises EGF + Noggin + R-spondin + Wnt-3a ***Highlighted in bold
are the compounds which showed the greatest improvement to the
culture medium.
[0711] In summary, the inventors have established long-term culture
conditions under which single crypts or stem cells derived from
murine small intestine (SI) expand over long periods of time.
Growing crypts undergo multiple crypt fission events, whilst
simultaneously generating villus-like epithelial domains in which
all differentiated cell types are present. The inventors have now
adapted the culture conditions to grow similar epithelial organoids
from mouse colon and human SI and colon. Based on the murine small
intestinal culture system, the inventors optimized the murine and
human colon culture system. They found that addition of Wnt3A to
the growth factor cocktail allowed mouse colon crypts to expand
indefinitely. Further addition of nicotinamide, a small molecule
Alk inhibitor and a p38 inhibitor was preferable for long-term
human SI and colon culture. The culture system also allowed growth
of murine Apc.sup.min adenomas, human colorectal cancer and human
esophageal metaplastic Barrett's epithelium. The culture technology
should be widely applicable as a research tool for infectious,
inflammatory and neoplastic pathologies of the human
gastrointestinal tract. Moreover, regenerative applications may
become feasible with ex vivo expanded intestinal epithelia.
Self-renewal of the small intestinal and colonic epithelium is
driven by the proliferation of stem cells and their progenitors
located in crypts. Although multiple culture systems have been
described (Evans G S et al. J Cell Sci 1992; 101 (Pt 1):219-31;
Fukamachi H. J Cell Sci 1992; 103 (Pt 2):511-9; Perreault N &
Jean-Francois B. Exp Cell Res 1996; 224:354-64; Whitehead R H et
al. Gastroenterology 1999; 117:858-65), only recently have
long-term culture systems become available that maintain basic
crypt physiology. Two different protocols were published which
allow long-term expansion of murine small intestinal epithelium.
Kuo and colleagues demonstrated long-term growth of small fragments
containing epithelial as well as stromal elements in a growth
factor-independent fashion (Ootani A et al. Nat Med 2009;
15:701-6). The inventors designed a culture system for single stem
cells by combining previously defined insights in the growth
requirements of intestinal epithelium. Wnt signalling is a pivotal
requirement for crypt proliferation (Korinek V et al. Nat Genet
1998; 19:379-83; Pinto D et al. Genes Dev 2003; 17:1709-13; Kuhnert
F et al. Proc Natl Acad Sci USA 2004; 101:266-71) and the Wnt
agonist R-spondin1 induces dramatic crypt hyperplasia in vivo (Kim
K A et al. Science 2005; 309:1256-9). Second, EGF signalling is
associated with intestinal proliferation (Dignass AU & Sturm A.
Eur J Gastroenterol Hepatol 2001; 13:763-70). Third, transgenic
expression of Noggin induces expansion of crypt numbers (Haramis A
P et al. Science 2004; 303:1684-6). Fourth, isolated intestinal
cells undergo anoikis outside the normal tissue context (Hofmann C
et al. Gastroenterology 2007; 132:587-600). Since laminin (.alpha.1
and .alpha.2) is enriched at the crypt base (Sasaki T et al. Exp
Cell Res 2002; 275:185-), the inventors explored laminin-rich
Matrigel to support intestinal epithelial growth. Matrigel-based
cultures have successfully been used for growth of mammary
epithelium (Stingl J et al. Breast Cancer Res Treat 2001;
67:93-109). Under this culture condition (R-spondin1, EGF, and
Noggin in Matrigel), the inventors obtained ever-expanding small
intestinal organoids, which displayed all hallmarks of the small
intestinal epithelium in terms of architecture, cell type
composition and self-renewal dynamics.
[0712] Despite extensive efforts, long-term adult human intestinal
epithelial cell culture has remained difficult. There have been
some long-term culture models, but these techniques and cell lines
have not gained wide acceptance, possibly as a result of inherent
technical difficulties in extracting and maintaining viable cells
(Rogler G et al. Scandinavian journal of gastroenterology 2001;
36:389-98; Buset M et al. In vitro cellular & developmental
biology: journal of the Tissue Culture Association 1987; 23:403-12;
Whitehead R H et al. In vitro cellular & developmental biology:
journal of the Tissue Culture Association 1987; 23:436-42; Deveney
C W et al. The Journal of surgical research 1996; 64:161-9; Pang G
et al. Gastroenterology 1996; 111:8-18; Latella G et al.
International journal of colorectal disease 1996; 11:76-83; Panja
A. Laboratory investigation; a journal of technical methods and
pathology 2000; 80:1473-5; Grossmann J et. al. European journal of
cell biology 2003; 82:262-70). Encouraged by the establishment of
murine small intestinal culture, the inventors aimed to adapt the
culture condition to mouse and human colonic epithelium. The
inventors now report the establishment of long-term culture
protocols for murine and human colonic epithelium, which can be
adapted to primary colonic adenoma/adenocarcinoma and Barrett's
esophagus.
Results
Establishment of a Mouse Colon Culture System
[0713] In an attempt to establish a mouse colon culture system, the
inventors explored our small intestinal culture condition (here
termed ENR: EGF+Noggin+R-spondin). In our experience, initial
growth of colon epithelium is often observed under the ENR culture
condition, but is invariably abortive. Organoid formation was
studied using epithelium isolated from the distal part of the mouse
colon. Under ENR conditions, the plating efficiency of single
distal colonic crypts was much lower than that of small intestine
(1-3% vs >90%) and these organoids could not be passaged.
Recently, the inventors have shown that Paneth cells produce
several Wnt ligands (Gregorieff A et al. Gastroenterology 2005;
129:626-38), and that the production of Wnt by these Paneth cells
is essential to maintain intestinal stem cells (Sato T et al.
Nature; 469:415-8). To determine the Wnt signalling status in colon
organoids, the inventors cultured colon crypts from Axin2-lacZ
mice, (a faithful Wnt reporter) (Lustig B et al. Mol Cell Biol
2002; 22:1184-93) or Lgr5-GFP knock-in mice (Lgr5 being a
Wnt-dependent stem cell marker)(Barker N et al. Nature 2007;
449:1003-7).
[0714] Freshly isolated colon crypts readily expressed Axin2-LacZ
or Lgr5-GFP at their bottoms, but they lost expression of the Wnt
reporters shortly after initiation of culture (FIG. 1a,b and FIG.
6). By contrast, small intestinal organoids constitutively
expressed the Wnt reporters at their budding structures (Sato T et
al. Nature; 469:415-8; Sato T et al. Nature 2009; 459:262-5). These
findings suggested that colon organoids produce insufficient
amounts of Wnt ligands to maintain colon stem cells. To overcome
this, the inventors added recombinant Wnt3a or Wnt3a-conditioned
medium to ENR culture medium (WENR medium). This increased plating
efficiency of crypts in the order of 10-fold. Colon crypts formed
organoids structures with numerous Axin2-LacZ (FIG. 1a) or
Lgr5-GFP+(FIG. 1b) buds, implying that Wnt activation was restored.
Freshly isolated colon crypts contain fully mature cells in their
upper parts, and the inventors reasoned that these mature cells may
interfere with organoid growth. When the inventors mildly digested
colon crypts into small clusters of cells, thus physically
separating proliferative crypt bottoms from differentiated upper
crypt regions, most of fragments derived from crypt top died, yet
cell clusters from colon crypt base efficiently formed organoids
(FIG. 1c).
[0715] Mouse small intestinal epithelium grown under ENR conditions
generates all differentiated epithelial cell types concomitant with
stem cell self-renewal. The inventors have shown previously that
the addition of Wnt3A to these cultures interferes with intestinal
differentiation and yields organoids that largely consist of
undifferentiated progenitors (Sato T et al. Nature; 469:415-8).
This is not unexpected given the central role of Wnt signalling in
the maintenance of the undifferentiated crypt progenitor state (van
de Wetering M et al. Cell 2002; 111:241-50). Consistent with this
observation, colonic organoids in WENR condition failed to
differentiate properly. Upon withdrawal of Wnt-3A, the inventors
observed differentiation along all epithelial lineages (FIG. 1d-f).
Of note, single sorted Lgr5+ colonic epithelial stem cells can form
organoids when cultured in the presence of Y-27632 for the first
two days.
Establishment of Human Colon Culture System
[0716] Encouraged by the success of the improved mouse colon crypt
culture, the inventors applied the culture condition to human colon
crypts. Although these crypts initially survived, most subsequently
disintegrated within 7 days. To increase the plating efficiency of
human colon crypts, the inventors screened candidate growth
factors, hormones and vitamins (list in FIG. 12).
[0717] Among these, the inventors found that gastrin and
nicotinamide (Precursor of NAD and found to suppress Sirtuin
activity (Denu J M. Trends Biochem Sci 2005; 30:479-83)) improved
culture efficiency (FIG. 12). The effect of gastrin on plating
efficiency was marginal. However, the hormone did not interfere
with intestinal differentiation and we decided to include gastrin
(hereafter shortened to `g`) in all human intestinal culture
conditions. Importantly, nicotinamide (10 mM) was essential for
prolongation of culture period beyond the initial 7 days (FIG. 2a).
Under this culture condition, human colonic organoids could be
expanded for at least 1 month. From 1 month onward, the colonic
organoids changed their morphology from budding organoids structure
into cystic structures (FIG. 2b left). Coinciding with the
morphological conversion, proliferation progressively decreased.
Occasionally, cystic organoids regained their proliferative
potential. However, all organoids eventually arrested growth within
3 months. A two-phase growth arrest has been observed in other
primary culture systems, such as mammary epithelial cells or
keratinocytes, and has been referred to as mortality stage 1 (M1;
senescence) and mortality stage 2 (M2; crisis) (Shay et al., 2006).
Multi-lineage differentiation was not observed in the human
intestinal organoids cultured in this condition even after the
withdrawal of Wnt (data not shown).
[0718] The inventors assumed that growth arrest occurred because of
inadequate culture conditions rather than a cell-intrinsic property
of senescence/replicative aging. The inventors therefore extended
our attempts to optimized the culture condition. The inventors
screened various small molecule modulators of MAP kinases, of
signaling molecules mutated in colon cancer, and of histone
modifiers (FIG. 12) under the WENR+gastrin+nicotinamide culture
condition. The inventors found that two small molecule inhibitors,
A83-01 (Alk4/5/7 inhibitor; nM) and SB202190 (p38 inhibitor; 10 uM)
significantly improved the plating efficiency. Other TGF-beta
receptor 1 (ALK 5) inhibitors that were also tested and showed the
same results as A83-01 were LY364947, SB431542, SB505124. It would
be expected that other ALK inhibitors would also work in the same
way. Furthermore, the combination of the two compounds
synergistically prolonged the culture period. The inventors
demonstrated that all of ten tested samples expanded for at least 6
months with weekly 1:5 split. Under this culture condition, the
human colonic organoids displayed budding organoid structures,
rather than the cystic structures seen under the previous culture
condition (FIG. 2b). The proliferating cells were confined to the
buds (FIG. 2c). Metaphase spreads of organoids more than 3 months
old consistently revealed 46 chromosomes in each cell (20 cells
each from three different donors; FIG. 2d). The inventors sequenced
the whole exome (all exons) of the colon organoids after two months
in culture. The number of mutations in the organoids was extremely
low. In fact in four parallel organoid cultures originating from
one clone, only one mutation was found which was present in all
cultures and therefore likely originated from the parental
tissue.
[0719] These results implied that Alk receptor and p38 signalling
negatively regulate long-term maintenance of human intestinal
epithelial cells. The inventors refer to the optimized culture
condition as the HISC (Human intestinal stem cell culture)
condition.
Human Intestinal Organoids Mimic In Vivo Differentiation
[0720] Under the HISC condition, the inventors failed to observe
differentiated cells. As was seen in the mouse colon organoids,
withdrawal of Wnt was required for mature enterocyte
differentiation in human organoids (FIG. 3a top panel and FIG. 7).
However, goblet and enteroendocrine cell differentiation remained
blocked. We found that Nicotinamide and SB202190 strongly inhibited
this differentiation, while withdrawal of the two reagents enabled
the organoids to produce mature goblet and enteroendocrine cells
(FIG. 3a (middle and bottom panel), 3b and FIG. 7. The same
differentiation inhibitory effects of Wnt, Nicotinamide and
SB202190 were observed in human small intestinal organoids.
Lysozyme+Paneth cells were observed in small intestinal organoids,
but not in colonic organoids (FIG. 3d). It has been reported that
p38 inhibitor treatment in vivo inhibits goblet cell
differentiation and increases intestinal epithelial proliferation
(Otsuka M. Gastroenterology 2010; 138:1255-65, 1265 el-9). Indeed,
the inventors observed the same phenotype in the p38 inhibitor
treated intestinal organoids (FIG. 3d vs. e). The inventors further
examined the response of human intestinal organoids to
Notch-inhibition. The inventors have previously shown that Notch
inhibition with either .gamma.-secretase inhibitors (dibenzazepine;
DBZ) or by conditional targeting of the Notch pathway transcription
factor CSL depleted intestinal stem cells, terminated intestinal
epithelial proliferation and induced goblet cell hyperplasia in
vivo (van Es J H et al. Nature 2005; 435:959-63). Indeed, upon
treatment with DBZ, the intestinal organoids ceased their
proliferation and most cells converted into goblet cells within 3
days (FIG. 3g vs f).
Establishment of APC-Deficient Adenoma and Colon Adenocarcinoma
[0721] Recently, the inventors reported efficient mouse intestinal
adenoma formation from Lgr5 stem cells in Lgr5-GFP-ires-CreERT2 x
APC.sup.flox/flox mice upon Tamoxifen-induced Cre activation
(Barker N et al. Genes Dev 2008; 22:1856-64). The inventors
isolated the intestinal adenomas 10 days after induction and
optimized the culture condition. The adenomas efficiently formed
cystic organoid structure without budding. Since APC loss
constitutively activates the Wnt pathway, the inventors expected
that R-spondin1 would become dispensable for adenoma organoid
growth. This was indeed observed. Furthermore, Noggin, which is
essential for long-term culture of normal small intestine, was
dispensable in adenoma organoids. Interestingly, the inventors
observed a loss of Lgr5-GFP but not Axin2-LacZ in adenomatous
organoids 7 days after withdrawal of Noggin (FIG. 4a,b and data not
shown). Similar observations were made for normal intestinal
organoids when grown in ER-medium (Sato T et al. Nature 2009;
459:262-5). This indicated that Noggin, most likely through
inhibition of BMP signals, is required to maintain Lgr5 expression,
but is not required for expansion of adenoma organoids. Freshly
isolated Lgr5.sup.hi (but not Lgr5.sup.low) cells isolated from
intestinal crypts can initiate organoid growth in vitro (Sato T et
al. Nature 2009; 459:262-5). To determine the existence of a
similar Lgr5-hierarchy within adenomas, the inventors isolated
Lgr5-GFP.sup.hi, GFP.sup.low and GFP.sup.-ve cells from EN-cultured
organoids and examined their organoid formation ability. After a 7
day culture, Lgr5-GFP.sup.hi showed the highest organoid-forming
efficiency. Yet, Lgr5-GFP.sup.low or.sup.-ve also formed organoids
with considerable efficiency (FIG. 4c). Of note, sorted GFP.sup.-ve
adenoma cells could give rise to Lgr5-GFP.sup.hi organoids ((FIG.
8)).
[0722] Many colorectal cancer cell lines have been isolated over
the past four decades. Typically, such cell lines emerge as rare,
clonal outgrowths after primary cultures of colon tumors enter
tissue-culture crisis. Currently, no robust culture system exists
which allows the consistent culture of primary human colon cancer
samples without culture crisis and the consequent clonal outgrowth
of culture-adapted cells. As a next step, the inventors applied
intestinal adenoma culture conditions to human colorectal cancer
samples. As expected, colon cancer cells required neither R-spondin
nor Noggin. EGF was dispensable in most colon cancer organoids,
while some colon cancer organoids decelerated their proliferation
after withdrawal of EGF. Distinct from mouse intestinal adenoma,
colorectal cancer organoids in the culture condition grew as
irregular compact structures rather than as simple cystic
structures (FIG. 4d).
[0723] The inventors examined the proliferation/differentiation
status of adenoma and colon cancer organoids. As expected, most of
cells were Ki67+. Consistent with the strong inhibitory effect of
Wnt on enterocyte differentiation (Figure if and FIG. 7), alkaline
phosphatase staining was not observed in both types of organoids
(FIG. 9). In contrast, we occasionally observed PAS+ goblet cells
and chromogranin A+ endocrine cells in adenoma organoids and in
some colon cancer organoids (FIG. 9).
Culturing Human Metaplastic Barrett's Epithelium
[0724] Barrett's Esophagus is marked by the presence of columnar
epithelium in the lower esophagus, replacing the normal squamous
cell epithelium as a result of metaplasia (Odze RD. Nat Rev
Gastroenterol Hepatol 2009; 6:478-90). The histological hallmark of
Barrett's Esophagus is the presence of intestinal goblet cells in
the esophagus. Exploiting the similarity between Barrett and
intestinal epithelium, the inventors subjected small Barrett's
epithelium (BE) biopsies to the human colon culture condition.
Under these culture conditions, normal esophageal squamous cells
transiently proliferated for 1 week, but the organoids could not be
passaged. Barrett's Esophagus epithelium could be maintained for up
to 1 month under HISC conditions (FIG. 5a). The BE organoids formed
cystic organoid structures indistinguishable from that of senescent
human colon organoids, and typically underwent growth arrest 1
month after the culture. Addition of FGF10 to the HISC condition
enabled the BE organoids to form budding structures and
significantly prolonged the culture duration (>3 months) (FIG. 5
b, c). In contrast to human intestinal organoids, BE organoids
remained Ki67+ with a minimal number of PAS+ and Mucin+ cells 4
days after withdrawal of Nicotinamide and SB202190. Treatment with
the .gamma.-secretase inhibitor DBZ (10 uM) for 4 days after the
withdrawal blocked proliferation and induced goblet cell
differentiation (FIG. 5 d-g). This supported our previous
suggestion that local delivery of such inhibitors may represent a
useful therapeutic strategy for the removal of Barrett's Esophagus
lesions by differentiation therapy (Menke V et. al. Disease models
& mechanisms 2010; 3:104-10). Of note, we occasionally observed
Lysozyme+Paneth cells (FIG. 10), which indicates that BE organoids
preserve multilineage differentiation.
Discussion
[0725] The protocols developed here allow robust and long-term
culture of primary human epithelial cells isolated from small
intestine, colon, adeno(carcino)mas and Barrett's Esophagus (table
3).
TABLE-US-00003 TABLE 3 List of components of the organoid culture
systems Reagent name Supplier Cat No. Solvent Stock solution Final
conc. Matrigel, GFR, phenol BD bioscience 356231 free Advanced
DMEM/F12 Invitrogen 12634-028 GlutaMAX-I Invitrogen 35050-079 200
mM 2 mM HEPES 1M Invitrogen 15630-056 10 mM Penicillin/Streptomycin
Invitrogen 15140-122 10000/10000 U/ml 100/100 U/ml N2 supplement
Invitrogen 17502-048 100x 1x B27 supplement Invitrogen 17504-044
50x 1x N-Acetylcysteine Sigma-Aldrich A9165-5G DW 500 mM = 81.5
mg/ml 1 mM EDTA Sigma-Aldrich 431788-25g DW 500 mM = 2 mM 14.6
g/100 ml Mouse recombinant Peprotech 250-38 100ug PBS/BSA 100 mg/ml
100 ng/ml noggin mouse recombinant Invitrogen PMG8043 PBS/BSA 500
mg/ml 50 ng/ml EGF human recombinant R- Nuvelo PBS/BSA 1 mg/ml 1
mg/ml spondin human recombinant Peprotech 100-26 PBS/BSA 100 mg/ml
100 ng/ml FGF10 mouse recombinant Millipore GF-160 PBS 10 mg/ml 100
ng/ml Wnt-3A Y-27632 Sigma-Aldrich Y0503 PBS 10 mM = 1 g/338 ml 10
mM A-83-01 Tocris 2939 DMSO 500 mM 500 nM SB202190 Sigma-Aldrich
S7067 DMSO 30 mM 10 mM Nicotinamide Sigma-Aldrich DW 1M 10 mM
[Leu15]-Gastrin I Sigma-Aldrich G9145 PBS/BSA 100 mM 10 nM DNase
Sigma-Aldrich DN25-1g PBS 200000 U/ml 2000 U/ml TrypLE express
Invitrogen 12605-036 Collagenase type XI Sigma-Aldrich C9407
Dispase Invitrogen 17105-041 70 um Cell strainer BD falcon 352350
All stock solutions and aliquoted Matrigel are stored in
-20.degree. C.
[0726] In contrast to murine small intestine, murine colonic
epithelial cells require Wnt ligand in the culture medium. The
inventors have previously reported that CD24.sup.hi Paneth cells
produce Wnt-3/11, which are essential for stem cell maintenance in
small intestine (Sato T, et al. Nature 2011; 469:415-8). Wnt-6 and
-9b mRNA are expresses at the bottom of colon crypts (Gregorieff A,
et al. Gastroenterology 2005; 129:626-38.). It remains undetermined
whether this local Wnt production by colon crypt base cells is
sufficient to activate canonical Wnt signal in vivo or there is
another source of Wnt ligand in colon mucosa. The difference
between human and mouse intestinal organoid culture conditions was
unexpectedly large. A83-01 inhibits ALK4/5/7, receptors that are
detected in both murine and human crypts by microarray. The
inventors are currently investigating the mechanism by which ALK
signal regulates human organoid growth. The inventors have not
observed cellular transformation in long-term cultures and no
chromosomal changes become obvious under the optimized culture
conditions. Furthermore, the organoids can undergo a considerably
higher number of cell division than reported for other adult human
epithelial culture system (Dey D et al. PloS one 2009; 4:e5329;
Garraway I P et al. The Prostate 2010; 70:491-501). It is generally
believed that somatic cells are inherently limited in their
proliferative capacity, a phenomenon called replicative aging
(Walen K H. In vitro cellular & developmental biology. Animal
2004; 40:150-8). Most normal human cells are believed to count the
number of times they have divided, eventually undergoing a growth
arrest termed cellular senescence. This process may be triggered by
the shortening of telomeres, and the consequent activation of DNA
damage signals (M1), or telomere attrition (M2). In the absence of
the two small molecule kinase inhibitors, human intestinal
organoids underwent growth arrest after 10-20 population doublings.
By contrast, the replicative capacity in the optimized culture
condition was extended at least up to 100 population doublings upon
addition of the inhibitors, which exceeded the Hayflick limit
(Hayflick L. The Journal of investigative dermatology 1979;
73:8-14). This result clearly indicates that the senescent
phenotype seen in the first culture system reflects inadequate
growth conditions, rather than inherent replicative aging.
[0727] The culture techniques can be used to study basic aspects of
stem cell biology and the control of differentiation, exemplified
by depletion of stem cells and goblet cell differentiation upon
Notch inhibitor treatment. Moreover, the organoid culture platform
may be used for pharmacological, toxicological or microbiological
studies on pathologies of the intestinal tract, as the organoids
represent more closely the intestinal epithelium than often-used
colon cancer cell lines such as CaCo2 or DLD 1. Lastly, since small
biopsies taken from adult donors can be expanded without any
apparent limit or genetic harm, the technology may serve to
generate transplantable epithelium for regenerative purposes.
EXAMPLE 2
Culturing Mouse Pancreatic Organoids
[0728] The use of a TGF-beta inhibitor was also tested in a culture
medium for mouse pancreatic organoids. The expansion medium that
was used was DMEM/F12 media (supplemented with P/S, Glutamax, 10 mM
Hepes, B27, N2 and N-Acetylcysteine), EGF (50 ng/ml), R-spondin
(10%), Noggin (100 ng/ml), FGF10 (100 ng/ml), A8301 (TGF-beta
inhibitor, 500 nM) and Gastrin (10 .mu.M). This differs slightly
from that of the above-described HISC culture used in Example 2 in
that there is no Wnt agonist (other than Rspondin) or Nicotinamide
and FGF10 is added. However, these culture media share a number of
key components (ENR+ gastrin+TGF-beta inhibitor), the addition of
the TGF-beta inhibitor being advantageous in both cases. Pancreas
organoids grown in these conditions could be expanded for >3
months and passaged at least 5 times.
[0729] Microarray experiments were carried out for the pancreas
organoids grown in the above-described expansion medium and the
results were compared to the adult pancreas, adult liver and
newborn liver (see FIG. 16A). The pancreas organoid clearly
clusters with the adult pancreas, rather than with the liver
samples, demonstrating a good phenotypic similarity with the adult
pancreas.
[0730] FIG. 16B shows the raw signal from the microarray experiment
comparing expression levels in pancreas organoids, adult pancreas,
adult liver and liver organoids for ductal markers, endocrine
markers and transcription factors necessary for Ngn3 expression
(Ngn3 is a transcription factor that is associated with the
specification of endocrine lineages). The high levels of expression
of Krt 19, Krt7 and other ductal markers in the pancreas organoids,
show that the pancreas organoids clearly have a ductal phenotype.
These pancreatic organoids were originally grown from ductal
preparations. The essential transcription factors for Ngn3
expression (Foxa2, Hnf6, Hnf1b, Sox9) were all also expressed in
the pancreas organoids, although expression of Ngn3 itself was not
detected under expansion conditions.
[0731] The expression levels of genes important for the generation
of insulin-producing cells are low. However, it is clear that in
the expansion medium, proliferation and expression patterns of the
pancreatic organoids closely resemble those seen in early
progenitor endocrine cells.
[0732] The pancreas is mainly formed by three different cell types:
acinar cells, ductal cells and endocrine cells. In a total RNA
sample of adult pancreas, 90% of the RNA comes from acinar cells,
so the expression levels of endocrine markers are very diluted in a
total pancreas sample. Therefore, further experiments are planned
for each specific cell type. For example, the inventors plan to
carry out a microarray comparison between pancreas organoids,
enriched acinar cell preparation, enriched ductal cell preparation
and enriched endocrine cell preparation, to have a better
estimation of the mRNA levels of the important genes in our
pancreas organoids compared with the levels present in insulin
producing cells. For example, in an enriched endocrine cell sample,
75-85% of the cells present would be insulin-secreting cells).
EXAMPLE 3
The Effect of Noggin on the Expansion Medium
[0733] To investigate the role of the BMP inhibitor, Noggin, in the
expansion medium, the inventors compared mRNA levels of early
endocrine markers and ductal markers in pancreatic organoids that
have always been cultured in EGFRA medium so have never been
cultured in the presence of Noggin with the level of expression of
the same markers in organoids that have always been cultured in
EGFRAN medium (i.e. always in the presence of Noggin). The
inventors also compared mRNA levels of these markers in pancreatic
organoids from which Noggin was added or removed from the cultures
respectively. Specifically, one sample of pancreatic organoids was
cultured in EGFRA medium and then Noggin was added and the
organoids were cultured for a further 2 or 4 days. Another sample
of pancreatic organoids was cultured in EGFRAN medium and then
Noggin was removed and the organoids were cultured for a further 2
or 4 days. The gene expression was compared and the results are
shown in FIG. 17A. It was found that Noggin reduces the expression
of keratin 7 and keratin 19 (ductal markers) showing that Noggin
blocks the differentiation towards the ductal phenotype (the
keratin levels in white and dark grey samples are lower than in the
black samples). Expression levels of some transcription factors
essential for the generation of insulin producing cells (i.e. Sox9,
Hnf6, Hnf1a, Pdx1, Nkx2.2, Nkx6.1 and Hnf1b) were unaffected by
Noggin. Although Noggin prevents the cultures from acquiring a full
ductal phenotype, which will likely prevent future differentiation
to insulin producing cells, the inventors include Noggin in the
expansion medium because it allows the cells to expand whilst
maintaining some ductal features in combination with features of
insulin-producing precursor cells.
[0734] The effect of the presence or absence of Noggin, or its
addition or withdrawal to EGFRA medium on Lgr5 gene expression was
assessed using pancreatic organoids obtained from pancreatic ducts.
The results in FIG. 17B show that pancreas organoids cultured with
Noggin express 2 fold more Lgr5 than pancreas organoids cultured
without Noggin (compare white bar second from left with black bar
on left). Addition (dark grey) or withdrawal (light grey) of Noggin
was also shown to affect Lgr5 levels. It is unclear whether the
increase in Lgr5 gene expression in the presence of Noggin is due
to an increased number of Lgr5+ cells or due to an increased level
of Lgr5 expression per cell. However, the present inventors show
here that BMP inhibitors, such as Noggin, promote expression of
Lgr5 and, therefore, result in more proliferative organoids. Thus,
BMP inhibitors are shown to be an advantageous component of the
expansion media.
[0735] This is surprising, because in the literature it is
described that BMP activity is useful for differentiation culture
of pancreatic cells. This conclusion is based on the observations
that BMP signalling is required for the differentiation into both
the ductal (see keratin7 and 19 expression) and endocrine cells.
Thus, the skilled person would expect the inclusion of a BMP
inhibitor, such as Noggin, to be disadvantageous in an expansion
medium. However, the inventors surprisingly found that the use of a
BMP inhibitor was advantageous because it resulted in more
proliferative organoids and higher expression of Lgr5.
EXAMPLE 4
Transplantation of Human Pancreatic Organoids Under the Kidney
Capsule in Mice
[0736] Pancreatic organoids, that had been expanded using the
protocol described in example 1 (see FIG. 18A), were transplanted
under the renal capsule of immunodeficient mice.
[0737] Just before transplantation, organoids were treated with
cell recovery solution (BD#354253, BD Biosciences) to get rid of
matrigel residues. Organoids were washed several times with PBS and
pelleted. Transplantation of these organoids under the renal
capsule of immunodeficient recipients was carried out using an NIH
recommended procedure for islet transplantation under the kidney
capsule ("Purified Human Pancreatic Islets, In Vivo Islets
Function", Document No. 3104, A04, Effective Date 7th Jul. 2008,
DAIT, NIAID, NIH). A week before the transplantation, hyperglycemia
was chemically induced in the recipient mice (NOD/SCID/IL2RgammaKO
a.k.a. NSG) with a high dose 130 mg/kg streptozotocin injection.
Blood glucose levels were monitored and mice having a blood glucose
above 18 mmol/1 were considered hyperglycemic.
[0738] For transplantion, the hyperglycemic recipient was
anesthetized and a small incision was made in the left flank to
expose the left kidney. Approximately 2.5-3.0 mm.sup.3 of organoids
were collected in a siliconized PESO transplantation tube and
transplanted under the kidney capsule using a Hamilton syringe.
After cauterizing the damaged capsule the kidney was placed back
into the abdominal cavity. The peritoneum and the skin were then
closed with 5-0 silk sutures.
[0739] One mouse was sacrificed three hours post-transplantation
and the graft was analyzed for mature beta cell and progenitor
markers. In this mouse, no insulin-producing cells could be seen in
the murine peri-renal capsule (FIG. 18B).
[0740] A further mouse was allowed to recover in the cage with a
heat pad, under close supervision. Bodyweights and blood glucose
levels of the transplanted mouse were monitored for 1 month. After
one month the mouse was sacrificed and the graft was analyzed for
mature beta cell and progenitor markers.
[0741] 1 month after transplantation, a number of insulin-producing
cells could be identified. These insulin-producing cells are all
the stained cells in FIG. 18C, a selection of which are circled for
enhanced clarity. In particular, insulin-positive cells appeared
from the ductal lining, whereas no insulin-positive cells were seen
in initial preparations.
[0742] The finding that the insulin producing cells are present 1
month after transplantation but are not present 3 hours after
transplantation demonstrates that the insulin producing cells
largely or only arise after transplantation.
[0743] These results show that cells taken from pancreatic
organoids of the present invention, cultured with the media and
methods of the present invention, can be transplanted into mice and
can promote the growth of insulin-producing cells in the pancreas.
Excitingly, human pancreatic organoids could be transplanted. This
opens a number of exciting possibilities for using transplanted
organoid cells to promote insulin production e.g. for treatment of
diabetes.
EXAMPLE 5
Liver Organoid Culture Comprising TGF-Beta Inhibitor
[0744] Under ER or ENRW conditions liver organoid cultures
self-renew, and can be maintained and expanded in a weekly basis,
for up to 1 year (FIG. 20A). The karyotypic analysis after 1 year
shows no evidence of chromosomal aberrations. More than 66% of the
cells analysed presented normal chromosomal counts and 13% of them
also showed polyploidy, a characteristic trait of hepatocytes (FIG.
20B).
[0745] The combination of EGF (50 ng/ml) and R-spondin 1 (1 ug/ml
supplemented with FGF10 (100 ng/ml), HGF (25-50 ng/ml) and
Nicotinamide (1-10 mM), were preferable for the long term
maintenance of the cultures. Under these conditions, we obtained
long-lived cell cultures that express biliary duct and some
hepatoblast or immature-hepatocyte markers (Glul, Albumine)
However, the number of cells positive for these hepatocyte markers
was very low. Under these culture conditions, no mature hepatocyte
markers (e.g. p450 Cytochromes) were detected. These results
suggest that the culture conditions described here facilitate the
expansion of liver progenitors able to generate hepatocyte-like
cells, albeit at lower numbers, but not fully mature hepatocytes
(FIG. 21A).
[0746] To enhance the hepatocytic nature of the cultures and obtain
mature hepatocytes in vitro, we first determined whether the three
supplemental factors (FGF10, HGF and Nicotinamide) added to EGF and
Rspondin1 were exerting either a positive or negative effect on the
hepatocyte expression, as well as on the self-renewal of the
culture. We generated liver organoid cultures and cultured them
either with EGF or EGF and Rspondin1 plus FGF10 or HGF or
Nicotinamide or the combination of these, and we split the cultures
once a week for a total period of 10 weeks. At each time-point we
also analysed the expression of several mature hepatocyte markers
(FAH, CYP3A11) and hepatoblast markers (albumin) (FIG. 21B).
[0747] It was observed that Rspondin1 and Nicotinamide combined
with FGF10 are essential for the growth and self-renewal of the
liver cultures (FIGS. 21C&D). Rspondin1 and Nicotinamide both
inhibit the expression of the mature marker CYP3A11 and yet promote
the expression of the hepatoblast marker albumin. The addition of
either FGF10 or HGF to media containing only EGF (without Rspondin1
and without nicotinamide), facilitated the expression of the mature
marker CYP3A11, albeit at very low levels (FIG. 21E). To identify
additional compounds that might facilitate hepatocyte
differentiation, we used two different approaches, both based upon
base conditions of: EGF+HGF and/or FGF10.
[0748] The first approach involved testing a series of compounds in
addition to the EGF+FGF10 or HGF condition. A complete list of the
compounds analysed is shown in table 4.
TABLE-US-00004 TABLE 4 Result Compounds Signal Concentration Alb
CYP3AII Exendin4 Glucagon like Sigma E7144 0.1-1uM peptide 2 analog
Retinoic Acid RAR-RXR receptor Sigma 25 nM ligand Retinoic Acid +
Exendin 4 Sonic Hedgehog Invitrogen 500-100 ng/ml C25II BMP4 BMP
signaling Peprotech 120- 20 ng/ml 05 DAPT Gamma-secretase Sigma
D5942 10 nM inhibitor A8301 Alk5/4/7 inhibitor Tocris 50 nM
Bioscience 2939 DAPT + A8301 +++ +++ FGF4 FGFR1, 2 ligand Peprotech
50 ng/ml FGF1 FGFR1, 2, 3, 4 ligand Peprotech 450- 100 ng/ml 33A
Dexamethasone Sigma D4902 10 .mu.M-1 mM 25MG Oncostatin M R&D
systems 10-1000 ng/ml (OSM) 495-MO-025 FGF4 + OSM + Dexa IGF
peprotech 100 ng/ml Valproic acid histone deacetylase Stemgent 04-
250 .mu.M inhibitor and 0007 regulator of ERK, PKC
wnt/.beta.-catenin pathways Sodium Butyrate histone deacetylase
Stemgent 04- 250 .mu.M inhibitor 0005 BIX01294 G9a HMTase Stemgent
04- 1 .mu.M inhibitor 0002 RG 108 DNA Stemgent 04- 1 .mu.M
methyltransferase 0001 inhibitor TSA 100 nM + - Hydrocortisone
glucocorticoid Sigma H6909 5 nM Oncostatin M R&D systems
10-1000 ng/ml (OSM) 495-MO-025 ARA Sigma A 0937 500 nM R 59022
Diacylglycerol Sigma D 5919 500 nM-50 nM + + kinase inhibitor
Arterenol andrenoreceptor sigma 500 nM-50 nM- bitrartre: -- agonist
A 0937 5 nM LIF 10.sup.3 PD 035901 MEK1 inhibitor Axon 500 nM
Medchem cat n 1386 CHIR99021 GSK3 inhibitor Axon 3uM Medchem cat n
1408 DMSO 1% L-Ascobic acid Sigma 1 mM 077K13021 VEGF Peprotech
Matrigel 50% Matrigel 20% VEGF + DEXA
[0749] The second approach took into account knowledge from
published developmental studies regarding the expression of the
transcription factors essential to achieve biliary and hepatocyte
differentiation in vivo. A comparative analysis of the expression
of transcription factors in the organoids under E or ER or ENRW
conditions supplemented with FGF10, HGF and Nicotinamide is shown
in FIG. 21. All the transcription factors required for Hepatocyte
specification were present, besides tbx3 and prox1. However, we
also noticed that the expression of specific biliary transcription
factors was highly upregulated in the cultures containing Rspondin1
(R), indicating that the culture gene expression was unbalanced
towards a more biliary cell fate.
[0750] Notch and TGF-beta signaling pathways have been implicated
in biliary cell fate in vivo. In fact, deletion of Rbpj (essential
to achieve active Notch signalling) results in abnormal
tubulogenesis (Zong Y. Development 2009) and the addition of TGFb
to liver explants facilitates the biliary differentiation in vitro
(Clotman F. Genes and Development 2005). Since both Notch and TGFb
signalling pathways were highly upregulated in the liver cultures
(FIG. 22) we reasoned that inhibition of biliary duct cell-fate
might trigger the differentiation of the cells towards a more
hepatocytic phenotype. A8301 was selected as an inhibitor of TGFb
receptor ALK5, 4, and 7 and DAPT as inhibitor of the
gamma-secretase, the active protease essential to activate the
Notch pathway. We first cultured the cells for 2 days in the
expansion conditions (ER media) and at day 2 (FIG. 23A) we started
the differentiation conditions by adding the combination of the
different compounds. Media was changed every other day, and the
expression of differentiated markers was analysed 8-9 days later.
The ER and ENRW conditions were used as negative control.
[0751] The combination of EGF+FGF10 with DAPT and A8301 resulted in
surprisingly large enhancement of expression of the hepatocyte
markers analysed (CYP3A11, TAT, Albumin) (FIG. 23B). The effect was
already detectable by day 5 and peaked at days 8-9 (FIG. 23C). The
maximal concentration efficiency was achieved at 10 uM (DAPT) and
50 nM (A8301) (FIG. 23D) respectively. The addition of
dexamethasone (a known hepatocyte differentiation molecule) did not
result in any improvement in gene expression. The combination of
EGF, FGF10, A8301 and DAPT not only enhances the expression but
also increases the number of hepatocyte-like cells, as assessed by
immunofluorescent against the hepatocyte markers albumin and 2F8,
and Xgal staining on AlbCreLacZ derived organoids (FIGS. 23E &
F). Therefore, we can conclude that the aforementioned
differentiation protocol facilitates the generation of
hepatocyte-like cells in vitro from liver stem cell cultures.
Methods
Reagents
[0752] Reagents used in the culture experiments are shown in Table
4. MiceLgr5-EGFP-ires-creERT2 mice (Barker N et al. Nature 2007;
449:1003-7), APC.sup.fl/fl (Sansom O J et al. Genes Dev 2004;
18:1385-90), Axing-lacZ mice (Lustig B et al. Mol Cell Biol 2002;
22:1184-93), C57B/6 wild type mice (6-12 week old) were genotyped
as previously described and were used for experiments.
Lgr5-EGFP-ires-creERT2 mice were crossed with APC.sup.fl/fl mice.
Cre enzyme activity was induced by intraperitoneal injections of
Tamoxifen (2 mg/mouse). The mice were euthanized 4 weeks after
Tamoxifen induction. Murine small intestines and colons were opened
longitudinally, washed with cold PBS and further processed for
crypt isolation. Regions containing intestinal adenomas were
identified using a stereomicroscope, cut out with a scalpel and
washed with cold PBS.
Human Tissue Materials
[0753] Surgically resected intestinal tissues were obtained from 30
patients from the Diaconessen Hospital Utrecht or the UMCU
Hospital.
[0754] Patient material was collected from 20 patients with colon
cancer (9 cecum-ascending colon, 7 sigmoid colon, 4 rectum; 33-86
years old), 5 patients with screening colonoscopy (33-63 years old)
and 5 patients with Barrett's esophagus (45-78 years old). For
normal tissue a distance of more than 3 cm to the tumors was kept.
The intestinal tissues were washed and stripped of the underlying
muscle layers. The tissue was chopped into around 5 mm pieces, and
further washed with cold PBS. Endoscopic biopsies (Intestinal or
esophageal) were obtained from the UMCU hospital. For each case, at
least 5 biopsy samples were collected and stored in cold PBS. This
study was approved by the ethical committee of DHU and UMCU, and
all samples were obtained with informed consent.
Crypt/Adenoma Isolation and Cell Dissociation
[0755] Intestinal fragments (murine normal colon, human normal
small intestine and colon) were further washed with cold PBS until
the supernatant was clear. Next, the tissue fragments were
incubated in 2 mM EDTA cold chelation buffer (distilled water with
5.6 mM Na2HPO4, 8.0 mM KH2PO4, 96.2 mM NaCL, 1.6 mM KCl, 43.4 mM
Sucrose, 54.9 mM D-Sorbitiol, 0.5 mM DL-Dithiothreitol) for 30 min
on ice (Gregorieff A Gastroenterology 2005(129)626-638). After
removal of the EDTA buffer, tissue fragments were vigorously
resuspended in cold chelation buffer using a 10-ml pipette to
isolate intestinal crypts. The tissue fragments were allowed to
settle down under normal gravity for 1 min and the supernatant was
removed for inspection by inverted microscopy. The
resuspension/sedimentation procedure was typically 6-8 times, and
the supernatants not containing crypts were discarded. The
supernatants containing crypts were collected in 50 ml-falcon tubes
coated with bovine serum albumin. Isolated crypts were pelleted,
washed with cold chelation buffer and centrifuged at 150-200 g for
3 min to separate crypts from single cells.
[0756] Murine colonic crypts were pelleted and resuspended with
TrypLE express (Invitrogen) and incubated for 15 min at 37.degree.
C. In this dissociation condition, colonic crypts were mildly
digested, thereby physically separating colonic crypt bottoms from
the top of the colon crypts. Intestinal fragments containing
adenomas from Tamoxifen-induced Lgr5-EGFPires-creERT2/APCfl/fl mice
were incubated in 2 mM EDTA chelation buffer for 60 min on ice.
Following washing with cold chelation buffer, most of the normal
intestinal epithelial cells were detached, while adenoma cells
remained attached to the mesenchyme. Next, the adenoma fragments
were incubated in digestion buffer (DMEM with 2.5% fetal bovine
serum, Penicillin/Stroptomycin (Invitrogen), 75 U/ml collagenase
type IX (Sigma), 125 .quadrature.g/ml dispase type II (Invitrogen))
for 30 min at 37.degree. C. The adenoma fragments were allowed to
settle down under normal gravity for 1 min and the supernatant was
collected in a 50 ml-falcon tube, pelleted and washed with PBS.
Isolated adenoma cells were centrifuged at 150-200 g for 3 min to
separate adenoma from single cells.
[0757] Biopsy samples from Barrett's epithelium and human colon
cancer samples, chopped into 5 mm pieces, were washed with PBS
several times. The tissue fragments were incubated in digestion
buffer for 60 min at 37.degree. C. After the digestion, tissue
fragments were manually picked under the microscope.
[0758] For sorting experiments, isolated crypts were dissociated
with TrypLE express (Invitrogen) including 2,000 U/ml DNase (Sigma)
for 60 min at 37.degree. C. Dissociated cells were passed through
20 .mu.m cell strainer (CellTrics) and washed with PBS. Viable
epithelial single cells were gated by forward scatter, side scatter
and pulse-width, and negative staining for propidium iodide or
7-ADD (eBioscience).
Culture of intestinal crypts, adenomas, Barrett's epithelium and
colon cancer
[0759] Isolated intestinal crypts, Barrett's epithelium and colon
cancer cells were counted using a hemocytometer. Crypts, fragments
of epithelium or single cells were embedded in matrigel on ice
(growth factor reduced, phenol red-free; BD bioscience) and seeded
in 48-well plates (500 crypts/fragments or 1000 single cells per 25
.mu.l of matrigel per well). The matrigel was polymerized for 10
min at 37.degree. C., and 250 .mu.l/well basal culture medium
(Advanced DMEM/F12 supplemented with penicillin/streptomycin, 10
min HEPES, Glutamax, 1.times.N2, 1.times.B27 (all from Invitrogen)
and 1 mM N-acetylcysteine (Sigma)) was overlaid containing the
following optimized growth factor combinations: murine EGF for
murine intestinal adenomas, ENR (murine EGF, murine noggin, human
R-spondin-1) for murine small intestinal crypts, WENR (recombinant
human Wnt-3A or Wnt-3A conditioned medium+ENR) for murine colonic
crypts, HISC (human intestinal stem cells:
WENR+gastrin+nicotinamide+A83-01+SB202190) for human small
intestinal/colonic crypts, HISC+human FGF10 for Barrett's
epithelium. Colon cancer cells show a heterogenous behaviour and
require either no addition of growth factors, murine EGF and/or
A83-01 and/or SB202190. For cell sorting experiments, Y-27632 (10
.mu.M; Sigma) was included in the medium for the first 2 days to
avoid anoikis. Reagents and concentrations of each growth factor
are indicated in FIG. 12. An overview of the optimized combinations
of growth factors and small molecule inhibitors for each organ is
given in FIG. 12.
Image Analysis
[0760] The images of organoids were taken by either confocal
microscopy with a Leica SP5, an inverted microscope (Nikon DM-IL)
or a stereomicroscope (Leica, MZ16-FA). For immunohistochemistry,
samples were fixed with 4% paraformaldehyde (PFA) for 1 h at room
temperature, and paraffin sections were processed with standard
techniques. Immunohistochemistry was performed as described
previously. For whole-mount immunostaining, crypt organoids were
isolated from Matrigel using Recovery solution (BD bioscience), and
fixed with 4% PFA, followed by permeabilization with 0.1% Triton
X-100. The primary antibodies were: mouse anti-Ki67 (1:250,
Monosan), rabbit anti-Muc2 (1:100, Santa Cruz), rabbit
anti-lysozyme (1:1,000, Dako), rabbit anti-synaptophysin (1:100,
Dako) and anti-chromogranin A (1:100, Santa Cruz). The secondary
antibodies were peroxidase-conjugated antibodies or
Alexa-568-conjugated antibodies.EdU staining followed the
manufacturer's protocol (Click-IT; Invitrogen). DNA was stained
with DAPI (Molecular Probes). Three-dimensional images were
acquired with confocal microscopy and reconstructed with Volocity
Software (Improvision).
Microarray analysis and Real-time PCR analysis
[0761] The data was deposited in the GEO database under the
accession number GSE28907.
EXAMPLE 6
Liver Organoid Culture Comprising Prostaglandin-2 or Arachidonic
Acid
[0762] In vitro survival, growth and expansion of liver organoids
was potently enhanced by addition of prostaglandin E2 (PGE2) or
Arachidonic acid (AA) to the basal medium.
[0763] FIGS. 25 and 26 show that addition of PGE2 at 50 nM (also
seen to work in the range 10-500 nM) or addition of AA at 10 ug/ml
(also works at 100 ug/ml, though not so well), results in a greater
number of larger organoids than using the basal medium alone.
Importantly, the addition of PGE2 or AA allows for a longer
expansion time. This means that organoids can be expanded for more
population doublings before there growth decreases or slows down.
Without PGE2 a growth reduction is seen after 5 weeks of culturing
at 5 fold expansion per week. With PGE2 there is no growth
reduction before at least 8 weeks at 5 fold expansion per week.
PGE2 was seen to have a slightly greater effect than AA. The basal
medium used was: hEGF (100 ng/ml, Invitrogen); human noggin
(hnoggin) (25 ng/ml, peprotech); gastrin (10 nM, sigma); hFGF10
(peprotech); nicotinamide (10 mM, sigma); A8301 (500 nM, Tocris);
hHGF (50 ng/ml, peprotech); Rspo conditioned media (10%).
[0764] PGE2 and AA are both in the same prostaglandin signalling
pathway (see FIG. 24), along with phospholipids, prostaglandin G2
(PGG2), prostaglandin F2 (PGF2), prostaglandin H2 (PGH2),
prostaglandin D2 (PGD2). It would be expected that addition of any
other activating component of this pathway would have the same
beneficial effect on the culture media.
[0765] Addition of PGE2 or AA is particularly beneficial for
expansion culture media. However, they may in some circumstances
also be included in differentiation media.
EXAMPLE 7
GSK3 Inhibitors are Effective Wnt Agonists in the Culture Media
[0766] CHIR99021, a GSK3 inhibitor, was shown to be an effective
Wnt agonist for the culture media. In particular, it was shown to
be a suitable replacement for Wnt in the culture media for colon
and liver organoids.
[0767] Furthermore, as an extension to Example 6, FIG. 25 shows
that human liver cells grown in the presence of both CHIR99021 (Wnt
agonist) and PGE2 result in more and larger organoids than cells
grown with either the Wnt agonist or PGE2 alone and certainly
more/larger organoids than in only the basal medium.
[0768] Therefore, GSK3 inhibitors could be used in the culture
media instead of, or in addition to, other Wnt agonists, such as
Wnt or Rspondin1-4.
[0769] It is surprising that CHIR99021 was such an effective Wnt
replacement because GSK3 is involved in a number of different
pathways, not only the Wnt pathway. This finding opens up the
possibility of designing other Wnt agonists targeting GSK3, which
might be useful in culture media.
EXAMPLE 8
Prostate
[0770] Isolation of Prostatic epithelium (Murine protocol).
[0771] The numbered steps correspond to FIG. 47.
i) Sacrifice male mouse at minimally 8 weeks of age to isolate a
mature prostate; isolate the urogenital sinus from the mouse. ii)
Remove seminal vesicles by breaking/cutting bloodvessels and
connective tissue and making a incision at the base at the urethra
iii) Remove the bladder by breaking/cutting it near the base at the
urethra iv) Remove remaining vesicles & fat tissue by gentle
tugging and cutting. What you should have left it the prostate
lobes (6 of them) and a pink structure in the middle, which is the
urethra; v) Remove urethra, easily recognized by the pink color
(stained dark in the picture). Carefully pull the prostate lobes,
so they are no longer attached to the urethra; isolate each lobe
individually, just by pulling them apart, or continue with the
whole prostate.
[0772] Next, mince the prostate (lobes) in small pieces; digest the
prostate in 1 ml 10 mg/ml Collagenase II (dissolved in ADMEM/F12)
for 11/2 hours at 37.degree. C.; after collagenase digestion only
"fingerlike" structures of epithelial cells should remain. [0773]
Wash in ADMEM/F12 [0774] Let the chunks settle down and draw off
supernatant (centrifugation at low speed gets rid of most the
mesenchyme) [0775] Centrifuge 50.times.G 5 min 4'C [0776] Resuspend
in 1 ml Trypsin (TLE) and digest for approximately 30 min 37'C.
Pipette up and down every 10 minutes to ensure digestion [0777]
Wash in ADMEM/F12 [0778] Either start culture in ENR or ENR+1 nM
DiHydroTestosterone (seed approximately 5000 cells per well) (0.1
nM-10 uM) we do not know an upper limit [0779] Or continue with
isolation of specific celltype via FACS
Results
[0780] Prostatic epithelial cells cultured in ENR+DiHydro
Testosterone, according to the methods described above, can be
maintained for 35 weeks so far. In the presence of testosterone,
the cultures expand the same as without testosterone. However, with
testosterone all cell types are present including stem cells,
transit amplifying cells and differentiated cells i.e. there is
increased differentiation whilst maintaining a stem cell
population. Prostate organoids grown in the presence of
testosterone also look more like the in vivo organ (see FIGS. 41
and 42). Furthermore, IHC and RT-PCR shows that prostate organoids
grown in the presence of testosterone contain both basal and
luminal cells.
[0781] The invention also provides the following numbered
embodiments: [0782] 1. A culture medium for expanding a population
of stem cells, wherein the culture medium comprises at least one or
more inhibitor that binds to and reduces the activity of one or
more serine/threonine protein kinase target selected from the group
comprising: TGF.beta. receptor kinase 1, ALK4, ALK5, ALK7, p38; and
wherein the culture medium allows continual growth for at least 3
months. [0783] 2. The culture medium according to embodiment 1,
wherein the at least one or more inhibitor comprises: [0784] a) an
inhibitor that binds to and reduces the activity of ALK5; and;
[0785] b) an inhibitor that binds to and reduces the activity of
p38. [0786] 3. The culture medium of embodiment 1 or embodiment 2,
wherein the inhibitor is an agent that binds to and reduces the
activity of its target by more than 95%; as assessed by a cellular
assay. [0787] 4. The culture medium of any one of the preceding
embodiments, wherein the inhibitor has an IC.sub.50 value of less
than 100 nM. [0788] 5. The culture medium of any one of the
preceding embodiments, wherein the inhibitor acts competitively;
non-competitively; uncompetitively; or by mixed inhibition. [0789]
6. The culture medium of any one of the preceding embodiments,
wherein the inhibitor acts competitively and binds to the
ATP-binding pocket of the serine-threonine protein kinase target.
[0790] 7. The culture medium of any one of the preceding
embodiments, wherein the inhibitor is: [0791] a) a small-molecule
inhibitor; b) a protein or peptide; c) an antisense
oligonucleotide; or d) an aptamer. [0792] 8. The culture medium of
any one of the preceding embodiments, wherein the small molecule
inhibitor has a molecular weight of between 50 and 800 Da. [0793]
9. The culture medium of any one of the preceding embodiments,
wherein the inhibitor is a pyridinylimidazole or a
2,4-disubstituted pteridine or a quinazoline-derived inhibitor.
[0794] 10. The culture medium of any one of the preceding
embodiments, wherein the inhibitor is added at a concentration of
between 10 nM and 10 .mu.M. [0795] 11. The culture medium of any
one of the preceding embodiments, wherein the inhibitor is selected
from the group of compounds comprising: SB-202190, SB-203580,
SB-206718, SB-227931, VX-702, VX-745, PD-169316, RO-4402257,
BIRB-796, A83-01, LY364947 SB-431542, SB-505124, SB-525334, LY
364947, SD-093, and SJN 2511. [0796] 12. The culture medium of any
one of the preceding embodiments, wherein SB-202190 or SB-203580 is
added to a concentration of between 50 nM and 100 uM. [0797] 13.
The culture medium according to any of the preceding embodiments,
wherein the stem cells are human stem cells. [0798] 14. The culture
medium according to any of the preceding embodiments, wherein the
stem cells are epithelial stem cells. [0799] 15. The culture medium
according to embodiment 14, wherein the human epithelial stem cells
are a) pancreatic stem cells; b) intestinal stem cells; or c) colon
stem cells. [0800] 16. The culture medium according to any of the
preceding embodiments, wherein the stem cells form part of an
organoid or isolated tissue fragment. [0801] 17. The culture medium
according to any of the preceding embodiments, wherein the stem
cells are cancer stem cells. [0802] 18. The culture medium of any
one of the preceding embodiments, wherein the percentage of cells
in the population of stem cells stem cells to have a normal
karyotype, after 1, 2 or 3 or more months, is more than 90%. [0803]
19. The culture medium of any one of the preceding embodiments,
wherein the percentage of cells in the population of stem cells
stem cells to have a normal phenotype, after 1, 2 or 3 or more
months, is more than 90%. [0804] 20. The culture medium of any one
of the preceding embodiments, wherein the stem cells survive for
more than three months; such as more than six months. [0805] 21.
The culture medium of any one of the preceding embodiments, wherein
the stem cells have an average population doubling time of 12 to 36
hours, of 18 to 30 hours, or of approximately 24 hours. [0806] 22.
The culture medium according to any of the preceding embodiments,
wherein the culture medium comprises a basal medium for animal or
human cells and: [0807] a) one or more bone morphogenetic protein
(BMP) inhibitor; [0808] b) one or more mitogenic growth factor; and
[0809] c) one or more Wnt agonist. [0810] 23. The culture medium
according to any of the preceding embodiments wherein, the culture
medium comprises gastrin and/or nicotinamide. [0811] 24. A method
for expanding a population of stem cells, wherein the method
comprises: [0812] a) providing a population of stem cells; [0813]
b) providing a culture medium according to any one of the preceding
embodiments; [0814] c) contacting the stem cells with the culture
medium; and d) culturing the cells under appropriate conditions.
[0815] 25. A composition comprising a culture medium according to
any of the preceding embodiments and stem cells. [0816] 26. A
composition comprising a culture medium according to the invention
and an extracellular matrix. [0817] 27. A culture medium supplement
comprising the one or more inhibitor according to any of the
preceding embodiments. [0818] 28. A hermetically-sealed vessel
containing a culture medium according to any of the preceding
embodiments or a culture medium supplement according to embodiment
27. [0819] 29. The culture medium according to any of embodiments 1
to 23 for the culture of Barrett's Esophagus epithelium, wherein
the culture medium further comprises FGF10. [0820] 30. Stem cells
or organoids obtained using the culture medium of any of the
preceding embodiments, for use in transplantation purposes or other
therapeutic applications. [0821] 31. A pancreatic organoid
comprising beta-cells. [0822] 32. The pancreatic organoid of
embodiment 31, further comprising .alpha. cells, .delta. cells and
PP cells. [0823] 33. The pancreatic organoid of any one of
embodiments 31 or 32, comprising .alpha. cells, .beta. cells,
.delta. cells and PP cells. [0824] 34. A pancreatic organoid of any
one of embodiments 31 or 33 that expresses one, two or all three of
Pdx1, Nkx2.2 and Nkx6.1. [0825] 35. A pancreatic organoid of any
one of embodiments 31 to 34 that expresses one, two or all three of
NeuroD, Pax6 and Mafa. [0826] 36. A pancreatic organoid of
embodiment 35 that additionally expresses Ngn3. [0827] 37. A
pancreatic organoid, for example a pancreatic organoid according to
any one of embodiments 31 to 36, which is capable of secreting
insulin following transplantation of the organoid into a patient.
[0828] 38. A pancreatic organoid as recited in any one of
embodiments 31 to 37 for use in treating a patient having an
insulin-deficiency disorder such as diabetes. [0829] 39. A method
of treating a patient having an insulin-deficiency disorder such as
diabetes comprising transplanting a pancreatic organoid according
to any one of embodiments 31 to 37 into the patient. [0830] 40. A
human organoid selected from the group consisting of a crypt-villus
organoid, a colon organoid, a pancreatic organoid, a gastric
organoid, a Barrett's Esophagus organoid, an adenocarcinoma
organoid and a colon carcinoma organoid. [0831] 41. A
small-intestinal or crypt-villus organoid, obtained using the
culture medium of any of embodiments 1 to 23, for use in treating
damaged epithelium, for example in microvillous inclusion disease
(MVID) patients. [0832] 42. A liver culture medium comprising or
consisting of a basal medium for animal or human cells to which is
added: one or more receptor tyrosine kinase ligand such as a
mitogenic growth factor (e.g. EGF), Nicotinamide, and preferably, a
Wnt agonist, preferably R-spondin 1-4 and/or CHIR99021 and one or
both of a) a prostaglandin pathway activator, such as PGE2 and/or
AA and b) a TGF-beta inhibitor such as A83-01.
Sequence CWU 1
1
30117PRTArtificial SequenceDSL peptide 1Cys Asp Asp Tyr Tyr Tyr Gly
Phe Gly Cys Asn Lys Phe Cys Arg Pro 1 5 10 15 Arg 222DNAArtificial
SequencePrimer 2gcagcattac ctgctctacg tt 22325DNAArtificial
SequenceReverse primer 3gcttgataag ctgatgctgt aattt
2548DNAArtificial SequenceProbe 4gcagccag 8518DNAArtificial
Sequenceforward primer 5catggaccgc ttcccata 18619DNAArtificial
Sequencereverse primer 6ggcacctgtc tgtccacat 1978DNAArtificial
SequenceProbe 7tggctctg 8818DNAArtificial SequenceForward primer
8cagccaacgc tgcttctc 18918DNAArtificial SequenceReverse primer
9tggcatggaa ttgacagc 18108DNAArtificial SequenceProbe 10cctcctgg
81121DNAArtificial SequenceForward primer 11ccgctactgg tgtaatgatg g
211222DNAArtificial SequenceReverse primer 12catcagcgat gttatcttgc
ag 22138DNAArtificial SequenceProbe 13aggagcag 81419DNAArtificial
SequenceForward primer 14gccagctcat caaggacag 191521DNAArtificial
SequenceReverse primer 15gcaggcatcg tagtagtgct g 21168DNAArtificial
SequenceProbe 16ttgcccag 81724DNAArtificial SequenceForward primer
17tgaccttgat ttattttgca tacc 241820DNAArtificial SequenceReverse
primer 18cgagcaagac gttcagtcct 201922DNAArtificial SequenceForward
primer 19gcttgccaca acttcctaag at 222022DNAArtificial
SequenceReverse primer 20tcagtttagt catggtggac ga
22218DNAArtificial SequenceProbe 21ggtggtgg 82218DNAArtificial
SequenceForward primer 22tgtggaaccg ggaagatg 182321DNAArtificial
SequenceReverse primer 23gaccacaggt atggttctgg a 21248DNAArtificial
SequenceProbe 24tggtggag 82521DNAArtificial SequenceForward primer
25cgatccagaa agatgatggt c 212619DNAArtificial SequenceReverse
primer 26cggaagcctc tgtctttcc 19278DNAArtificial SequenceProbe
27ggatggag 82819DNAArtificial SequenceForward primer 28tcctcctcag
accgctttt 192921DNAArtificial SequenceReverse primer 29cctggttcat
catcgctaat c 21308DNAArtificial SequenceProbe 30actcccag 8
* * * * *
References